Solution based DNA circularization detection of pathogens

Abstract

The present invention relates to a method for determining whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases. The invention also relates to kits of parts for performing such methods and uses thereof.

Description

[0001] PV ref: 84489PC01

[0002] 1

[0003] SOLUTION BASED DNA CIRCULARIZATION DETECTION OF PATHOGENS

[0004] Technical field of the invention

[0005] The present invention relates in particular to a method of detecting Mycobacterium spp. in a sample using DNA circularization by Mycobacterium spp. DNA Topoisomerase I enzyme as an indicator. In particular, the present invention relates to solution-based system for the circularization step and direct incorporation of labelled nucleotides during RCA amplification.

[0006] Background of the invention

[0007] Tuberculosis (TB) is a serious infectious disease affecting health worldwide. TB is caused by members of the Mycobacterium tuberculosis complex (Mtb) and is one of the leading causes of death caused by a single infectious agent. Even though TB is preventable and curable, it remains a threat to global health, as diagnosis and treatment are not always accessible to everyone. Moreover, there is an increase in the development of antimicrobial resistant mycobacteria, which are currently responsible for a quarter of the deaths caused by TB. According to the latest WHO report for 2020, an estimated number of 10 million people were infected with TB and around 1.5 million died. Furthermore, WHO estimates that one quarter of the world is infected with TB, primarily with a latent infection, which in around 10% will progress to active TB. TB is most prevalent in South- East Asia, Africa and the Western Pacific and continues to afflict many countries due to inadequate health care systems and lack of diagnosis caused by e.g. initial mild symptoms of TB, economics and logistics. Of the 10 million people estimated to be infected with TB only 5.8 million were diagnosed in 2020. This ultimately results in under-treatment, increased spread of the disease and more deaths. Hence, improving TB diagnostics is an important tool to decrease the large burden of TB worldwide.

[0008] Accurate diagnosis of TB is troublesome as a cause of slow-growing mycobacteria, requirement of difficult obtainable test material from the lungs (called sputum) and the low resources in high TB burden areas. Diagnosis is currently based on primarily sputum smear microscopy and the GeneXpert polymerase chain reaction (PCR) amplification test. The gold standard and most sensitive method is PV ref: 84489PC01

[0009] 2 cultivation of the mycobacteria. This can, however, take from 2-8 weeks and is not convenient to use for first line diagnostics. Sputum smear microscopy remains one of the most used methods in low-resource areas as it is rapid, simple and inexpensive, but the method suffers from low sensitivity and specificity and difficulties in obtaining sputum samples from some patients. An alternative is the WHO endorsed GeneXpert MTB / RIF PCR test from Cepheid, which is simple, fast and has a sensitivity and specificity of 85% and 98% in high resource settings. Moreover, the method can detect antimicrobial resistance towards the most used treatments. The method does, however, require a specialized expensive PCR machine and the cost per test is high (> 15$). Moreover, the existing techniques require sputum of good quality as test material. Sputum can be troublesome to work with and difficult to obtain from children and patients already diagnosed with HIV. There is a need for fast, simple and cheap tests, which are point-of-care friendly, and do not require trained staff and stable electricity. More importantly, the tests should be based on non-invasive samples (such as saliva) and not on sputum to improve diagnosis of i.e. children.

[0010] WO2024 / 094858 Al discloses a method for detecting TB in saliva, wherein a circularizable oligonucleotide is attached to a solid support before being brought in contact with a saliva sample. WO2024 / 094858 Al is silent in respect of circularization in solution, when labelled nucleotides are incorporated.

[0011] WO2008 / 148392 Al discloses enzyme activity assay using rolling circle amplification for verifying that a sample contains the enzyme activity in question, such as human topoisomerase 1.

[0012] Franch et al. (Nanoscale, 2019, 11, 587) discloses a DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. In the supplementary data section, it is disclosed that circularization in solution does not work (supplementary figure 1 panel B). PV ref: 84489PC01

[0013] 3

[0014] WO 2013 / 029631 A2 discloses microfluidic-implemented methods of detecting a DNA-modifying enzyme. In WO 2013 / 029631 A2 the cleavage and ligation steps are separated, and the circularization step takes place when coupled to a surface.

[0015] Hence, an improved method of detecting TB would be advantageous, and, in particular, a more efficient and / or reliable method of detecting TB in saliva would be advantageous.

[0016] Summary of the invention

[0017] In the present invention, synthetic DNA circles are generated by a pathogen e.g. Mycobacteria spp. TOPI in solution, whereafter the circles are hybridized to a primer-anchored surface to support RCA or RCA is made in solution, where incorporation of e.g. biotinylated nucleotides occurs. After RCA, e.g. HRP conjugated with an anti-biotin antibody or streptavidin is bound to the RCA product, and signals may be generated by adding ECL (for chemiluminescence readout) or TMB (for colorimetric readout).

[0018] Surprisingly, readout only works if the circles are made in solution. Another surprising result is that it has been achieved to make the detection work in a lateral flow device, wherein the RCA product comprising labelled nucleotides is detected.

[0019] Example 1 illustrates two different ways the method of the invention can be performed, namely circularization in solution followed by RCA amplification on a solid support (figure 1A) and circularization in solution followed by RCA amplification in solution and finally a lateral flow readout (figure lb).

[0020] Example 2 shows that the solid support assay for detection of mycobacterium (mTOPl) could not be adapted to an ECL based readout based on the coupling of anti-biotin-antibody HRP to biotinylated RCA products.

[0021] Example 3 shows that unexpectedly the incubation of mTOPl with the substrate in solution works well for an ECL-based readout.

[0022] Example 4 demonstrates that exonuclease treatment of the circle mixture before RCA improves the readout for some sequences and not for others.

[0023] Example 5 shows that 40 pmol of DNA substrate SEQ ID NO: 1 is optimal concerning signal-to-noise. PV ref: 84489PC01

[0024] 4

[0025] Example 6 demonstrates that increasing the concentration of MgCh and omitting MnCh in the reaction buffer gives an increased signal.

[0026] Example 7 shows that the assay of the invention allows for quantitative detection of M. smegmatis with a detection limit of 1000 CFU / |JL.

[0027] Example 8 shows that the assay of the invention allows for detection of Mtb in saliva and in sputum from TB positive individuals.

[0028] Example 9 shows that Plasmodium Topoisomerase 1 (pTOPl) can be detected using the assay of the invention, thus also allowing for detection of malaria. Example 10 shows detection of Plasmodium infection in saliva from confirmed malaria-positive individuals.

[0029] Example 11 shows that the assay of the invention allows for detection of mpoxTOPl in saliva, thus allowing for detection of mpox.

[0030] Example 12 illustrates the lateral flow format of the invention.

[0031] Examples 13-15 shows that the lateral flow format can indeed work for RCA products in different formats. The lateral flow format can also be optimized for different types of membranes.

[0032] Example 16 illustrates optimizations of the lateral flow dipstick format.

[0033] Example 17 shows that decreasing the NaCI concentration in the circle reaction increases the signal from saliva collected from a TB-positive individual and decreases the background signal obtained from saliva collected from a TB- negative individual.

[0034] Example 18 shows that the method of the invention works in matching sputum, saliva and tongue swabs from confirmed tuberculosis positive or negative individuals.

[0035] Example 19 confirms that it is possible to detect malaria in saliva using the method of the invention. Example 19 also describes the use of a branching oligonucleotide during RCA.

[0036] Hence, advantages of the present invention compared to related technologies are at least the following:

[0037] - Incorporation of labelled oligonucleotides during RCA and subsequent detection is faster than hybridization of a labelled oligonucleotide after RCA.

[0038] - The method is easier to use, since fewer steps and fewer components are required. PV ref: 84489PC01

[0039] 5

[0040] Different types of pathogens may be detected.

[0041] - The assay is functional in a lateral flow setup, wherein the RCA products are detected.

[0042] Lower background signal, since surplus of substrate can be removed, such as by exonuclease treatment or filtration.

[0043] Thus, an object of the present invention relates to the provision of an improved method to detect pathogens, such as Mycobacterium spp., which expresses DNA manipulating enzymes such as topoisomerases.

[0044] In particular, it is an object of the present invention to provide a method to detect Mycobacterium spp. that solves the above-mentioned problems of the prior art with respect to cost and speed.

[0045] Thus, one aspect of the invention relates to a method for determining whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases, the method comprising a) having a sample, such as a saliva sample or tongue swab, provided; b) providing a single stranded DNA oligonucleotide (1) comprising:

[0046] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl) (2);

[0047] - a primer-annealing element, complementary to a DNA oligonucleotide (4); and c) incubating in solution, preferably in the presence of Mg2+ions, the biological sample of step a) with the oligonucleotide (1) from step b), wherein if the DNA modifying enzyme, such as mTOPl (2) is present in the sample from step a), the single stranded DNA oligonucleotide of step b) is circularized (3); d) hybridizing the oligonucleotide (1) from step c) to the DNA oligonucleotide (4); PV ref: 84489PC01

[0048] 6 e) determining the level of circularized oligonucleotides in the sample from step c) using Rolling Circle Amplification (RCA) with incorporation of labelled nucleotides, preferably the label is a first capture moiety, such a biotin moiety, such as biotin-dCTP; f) comparing said determined level to a reference level; and g) determining that if said level is above said reference level it is indicative of the sample comprising the pathogen; or if said reference level is equal to or below said reference, it is indicative of the sample not comprising the pathogen.

[0049] In a preferred embodiment, the pathogen is Mycobacterium spp, preferably from the Mycobacterium tuberculosis complex.

[0050] Another aspect of the present invention relates to a kit of parts comprising a) a single stranded DNA oligonucleotide (1) comprising:

[0051] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl) (2);

[0052] - a primer-annealing element, complementary to a DNA oligonucleotide (4); b) a labelled DNA oligonucleotide (4), suitable for acting as a primer for an RCA reaction of a circularized version of the single stranded DNA oligonucleotide (1); and c) a lateral flow device adapted to detect an RCA product comprising labelled nucleotides, such as biotins; d) optionally reaction components, such as a lysis buffer, exonuclease mix, RCA mix and / or detection mix; e) optionally, a gel filtration column, adapted for removing excess reagents; and f) optionally a branching oligo, such as SEQ ID NO: 17.

[0053] Yet a further aspect of the invention relates to the use of the kit of parts according to the invention for determining, whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, PV ref: 84489PC01

[0054] 7 said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases.

[0055] In an embodiment, the use is for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with Mycobacterium spp, preferably the Mycobacterium tuberculosis complex.

[0056] Brief description of the figures

[0057] Figure 1

[0058] Figure 1 shows a schematic illustration of the SOLEAD (SOLution based Enzyme Activity Detection) protocol. A) SOLEAD combined with ECL readout from solid support. The TB-causing Mtb complex (mycobacterial species causing TB in humans) present in saliva is lysed to release the target enzyme mTOPl, which reacts with a specific DNA sequence to generate circles. This step is done in solution. Thereafter the circles are hybridized to a solid support anchored primer and phi29 polymerase is added to generate rolling circle amplification of the circle in the presence of biotin-conjugated dCTP. This generates RCA products with biotins that can bind HRP functionalized with anti-biotin antibodies. Readout is achieved by adding ECL that is converted to a chemiluminescent product by HRP. Alternatively, TMB that is converted to a blue-colored product by HRP can be used for readout. B) SOLEAD combined with lateral flow readout. Mtb is lysed and circles are generated as described above. RCA is performed with a FAM end- labelled primer (SEQ ID NO: 15) in the presence of biotin-conjugated dCTP to generate RCA products with dual FAM and biotin labeling. The products are applied to a lateral flow device and captured on the test line by anti-FITCH antibodies and visualized by labeling using gold nanoparticles conjugated with an anti-biotin antibody.

[0059] Figure 2

[0060] Figure 2 shows SOLEAD protocol on a solid support. A) Depiction of representative images of the chemiluminescent signals obtained by using the SOLEAD protocol on a solid support in the absence (-) or in the presence (+) of the msTOPl enzyme. B) Graphical representation of the results of three independent experiments. PV ref: 84489PC01

[0061] 8

[0062] Figure 3

[0063] Figure 3 shows SOLEAD protocol with circularization in solution and by using different oligonucleotide sequences. Depiction of the representative images of the chemiluminescent signals obtained by using the SOLEAD protocol with circularization in solution in the absence (-) or presence (+) of msTOPl. The SEQ ID NOs: 2, 3, 4, 5 and 6 used in the experiment is shown to the right for the picture.

[0064] Figure 4

[0065] Figure 4 shows the effect of exonuclease digestion of the circles generated by the SOLEAD protocol with circularization in solution. The SOLEAD protocol was performed by incubation of SEQ ID NO: 1 (40 pmol) or SEQ ID NO: 6 (40 pmol) with M. smegmatis extract from 40000 CFU spiked-in saliva or saliva alone as a negative control (background signal shown by dotted line). The bars represent the ratio between signals obtained when analyzing M. smegmatis extract spiked-in saliva and signals obtained when analyzing saliva alone. + : circles digested with exonucleases prior hybridization to the solid support; -: circles not digested with exonucleases prior hybridization to the solid support. The results are plotted as the mean + / - standard deviation.

[0066] Figure 5

[0067] Assessment of optimal oligonucleotide sequence concentration. Graphical representation of quantification analysis of two experiments performed using the SOLEAD protocol with an oligonucleotide sequence SEQ ID NO: 1 titration and by a fluorescence microscopy readout (to obtain optimal quantitative results). The experiments were performed by using M. smegmatis extract spiked-in saliva or saliva alone. Circles: lOpmol; Squares: 20 pmol; Triangles: 40 pmol; Diamonds: 80 pmol.

[0068] Figure 6

[0069] Optimizations of buffers conditions for circularization in the SOLEAD protocol. Two experiments were performed by using M. Smegmatis extract (40000 CFU / / zL) spiked-in saliva or saliva alone. For each experiment, 2 pellets of M. Smegmatis were used, and 2 repetitions were made for each condition for each pellet. The analysis was performed using the SOLEAD protocol with a fluorescence microscopy readout and the signals counted in the microscope were normalized to the signals obtained with samples assayed in 200 mM NaCI, 10 mM MgCh and 10 PV ref: 84489PC01

[0070] 9

[0071] MnCh, previously reported as the standard protocol. White circles: 2 technical repetitions from one extract; Black circles: 2 technical repetitions from another extract. Results are depicted as average + / - SEM.

[0072] Figure 7

[0073] SOLEAD protocol detection limit using chemiluminescent readout to analyze M. smegmatis extracts. Graphical depiction of the intensity of chemiluminescence signals obtained when measuring the activity of mTOPl enzyme extracted from M. smegmatis bacteria ranging from 40000 to 5000 CFU / ul and spiked-in saliva. Each dot represents one single circularization sample. ****P<0.0001; * P<0.04.

[0074] Figure 8

[0075] Test of clinical samples from 8 TB and HIV positive individuals A) Depiction of the representative images of the chemiluminescent signals obtained by using the SOLEAD protocol with circularization in solution in the presence of extracts from 4 saliva and 4 sputum samples from TB-positive individuals. +: positive control, circles obtained by using M. smegmatis extract; -: saliva from two negative individuals, collected in Denmark. B) Graphical depiction of the intensity of chemiluminescence signals visualized in A. Dark grey bars: saliva; Light grey bars: sputum. Black bar: positive control with ms extract circles; White bar: Saliva from 2 TB negative individuals (collected in Denmark).

[0076] Figure 9

[0077] Detection of pTOPl with the SOLEAD protocol with chemiluminescence readout. Graphical depiction of the signal intensity obtained when measuring the activity of pTOPl enzyme by using the SOLEAD protocol with ECL readout in the presence or absence of exonucleases digestion (Exo).

[0078] Figure 10

[0079] Detection of Plasmodium in clinical saliva samples. A) Left panel), graphical depiction of chemiluminescence SOLEAD results obtained when measuring extracts from two saliva samples from confirmed malaria positives and two saliva samples from presumed negative individuals prepared by 2-5 times vortex with glass beads. The average of the results from two individual experiments is shown by vertical lines. A) Right panel) raw data obtained with a CCD camera to capture the chemiluminescent signal. B) The results obtained by analyzing 30 saliva samples from confirmed malaria-positive individuals and 31 saliva samples from presumed malaria-negatives using the SOLEAD protocol with PV ref: 84489PC01

[0080] 10 chemiluminescence readout. The average of the results is shown by vertical lines. To compensate for slide-to-slide variations, the chemiluminescence SOLEAD signals were normalized to the average of the negative samples vortexed 2 times (A) or to the average of the negative samples (B).

[0081] Figure 11

[0082] Detection of mpoxTOPl in saliva. Serial dilutions of purified mpoxTOPl was incubated with 5 pmol of mpoxTOPl specific SEQ ID NO: 10 in the presence of saliva. The circularization, RCA and visualization was performed as described in example 4. Figure 11.

[0083] Figure 12

[0084] Sequential method for sample testing in a generic 96 well plate. Half dipsticks were assessed through the sequential insertion of the strip into sample, colloidal gold conjugate and dilution buffer as shown, where running buffer comprised phosphate buffered saline with Tween 20.

[0085] Figure 13

[0086] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-biotin test line using sequential method (anti-biotin gold conjugate). The bars represent the average visual test score for each sample and type of nitrocellulose membrane. The visual test score was measured by two operators against a printed reference card, and average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0087] Figure 14

[0088] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-FITC test line using sequential method (anti-biotin gold conjugate). The bars represent the average visual test score of each sample and type of nitrocellulose membrane. The visual test score was measured by two operators against a printed reference card, and the average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0089] Figure 15

[0090] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-Biotin test line using sequential method and ODO.5 antibiotin conjugate gold. The bars represent the average visual test score of each sample and type of nitrocellulose membrane. The visual test score was measured PV ref: 84489PC01

[0091] 11 by two operators against a printed reference card, and the average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0092] Figure 16

[0093] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-FITC test line using sequential method and ODO.5 antibiotin conjugate gold. The bars present the average visual test score of each sample and type of nitrocellulose membrane. The visual test score was measured by two operators against a printed reference card, and the average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0094] Figure 17

[0095] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-biotin test line using combination method (ODO.5 antibiotin conjugate gold). The bars represent the average visual test score of each sample and type of nitrocellulose membrane. The visual test score was measured by two operators against a printed reference card, and the average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0096] Figure 18

[0097] Comparison of nitrocellulose membranes tested with rolling circle product samples on half dipsticks with anti-FITC test line using combination method (ODO.5 antibiotin conjugate gold). The bars represent the average visual test score of each sample and type of NC membrane. The visual test score was measured by two operators against a printed reference card, and the average of two replicates performed. The results are plotted as the mean + / - standard deviation.

[0098] Figure 19

[0099] Diagram showing sandwich assay format. If the RCPs incorporate both a FAM and biotin probe a sandwich format could be used. A lateral flow format with an anti- FITC or anti-biotin test line with an anti-FITC or anti-biotin conjugate could be evaluated. If the RCP contains either a biotin probe or FAM probe a single sandwich and T-line system would be required. By adding the sample to the test strip ahead of the anti-biotin conjugate any excess biotin in the RCP solution would not interfere with the result. PV ref: 84489PC01

[0100] 12

[0101] Figure 20

[0102] Artwork-Abingdon Health Generic Scorecard-Gold. Printed reference card used for the visual scoring of all devices tested.

[0103] Figure 21

[0104] Detection of FAM and biotin-labelled mTOPl or pTOPl-specific RCA products on the half dipstick lateral flow format using a design with anti-FITC functionalized test line and anti-biotin antibody gold-conjugated dye. #40, negative sample composed of circularized SEQ ID NO: 1. #36, positive sample composed of RCA products made from circularized SEQ ID NO: 1. #42, negative sample composed of circularized SEQ ID NO: 7. #43, positive sample composed of RCA products made from circularized SEQ ID NO: 7. The scoring of the test line staining in comparison to a standard scorecard is shown to the left of the pictures of the dipsticks.

[0105] Figure 22

[0106] Results of testing a saliva sample from a TB-positive individual (51) and a TB- negative individual (6) using different NaCI concentrations during circularization and exonuclease treatment (as shown to the left). The scoring of the test line staining in comparison to a standard scorecard is shown to the left of the pictures of the dipsticks.

[0107] Figure 23

[0108] Validation of Pasodect of tuberculosis detection in Guinea Bissau. Top: Picture of typical result on the LF sticks. Bottom: Graphical depiction of the results from a validation study performed in Bissau. Samples from positive and negative individuals were collected and analysed using Pasodect in combination with lateral flow readout. The score is depicted on the Y-axis.

[0109] Figure 24

[0110] Validation of Pasodect for detection of malaria in saliva from confirmed positive individuals in comparison to malaria negative individuals. Saliva samples from positive or negative individuals stored at 4°C were analyzed by Pasodect in combination with lateral flow readout. The score is depicted on the Y-axis. As controls samples with or without spike-in purified pTOPl was used. PV ref: 84489PC01

[0111] 13

[0112] The present invention will now be described in more detail in the following.

[0113] Detailed description of the invention

[0114] Definitions

[0115] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0116] Oligonucleotide

[0117] In the present context, an oligonucleotide is a sequence of DNA (or RIMA) nucleotide residues that form a molecule. Oligonucleotides can bind their complementary sequences to form duplexes (double-stranded fragments) or even fragments of a higher order. Oligonucleotides can be on a linear form, but also exist as circular oligonucleotide molecules, such as single stranded circular DNAs. When referring the length of a sequence, reference may be made to the number of nucleotide units or to the number of bases. Furthermore, the typical DNA or RNA nucleotides may be replaced by nucleotides analogues such as 2'-O-Me-RNA monomers, 2'-O- alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, locked nucleic acid (LNA) monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, 1,5-anhydrohexitol nucleic acid (HNA) monomers, peptide nucleic acid (PNA), and morpholinos. In the present context, the oligonucleotide (1) should be an enzymatic target for Mycobacterium tuberculosis DNA Topoisomerase I enzyme and enable subsequent amplification by e.g. RCA. Thus, preferably the oligonucleotide is DNA, but may comprise one or more modified nucleotides. Also preferred is the one or more single stranded oligonucleotides (1) free of modified nucleotides. In the example section, nonmodified single stranded DNA oligonucleotides (1) have been used except for 5'- or 3'- end labeling as indicated.

[0118] Incubating in solution

[0119] In the present context the term "incubating in solution", refers to the oligonucleotide (1) being in solution when incubated with the sample in question. Hence, the oligonucleotide (1) is not hybridized or linked to a primer coupled to a solid support, such as a glass slide, when incubated with the sample in question. PV ref: 84489PC01

[0120] 14

[0121] Topoisomerase IA:

[0122] Type IA topoisomerases create a single break in DNA and pass a second strand or duplex through the break. This strand passage mechanism shares several features with type IIA topoisomerases. They both form a 5' phosphotyrosine intermediate and require a divalent metal ion to perform its work. Unlike type II topoisomerases, type IA topoisomerases do not use energy to do its work (with the exception of reverse gyrase). Like all known type IA topoisomerases, mycobacteria TOPIA regulates DNA topology in a Mg2+dependent reaction that involves temporary nicking of the DNA and the formation of a transient covalent 5'-phosphotyrosyl cleavage complex. Restoration of intact DNA and release of the enzyme is ensured by subsequent ligation of the 3'-OH DNA end formed during cleavage.

[0123] Chemiluminescence assay

[0124] In the context of the present invention, the term "chemiluminescence assay" refers to a readout wherein a signal is generated by emission of light from a chemical reaction. Typically, luminol or its derivatives act as a substrate, which undergo multiple oxidation reactions to form a product in the excited state, while returning to ground state emit light in the shorter wavelength (425 nm). Enhanced chemiluminescence (ECL) substrates, are Horseradish Peroxidase (HRP) substrates that are typically two-component systems, containing luminophores and co-reactants, which are added to the system to produce light through an electrochemical reaction.

[0125] Luminophores: These are the light-emitting molecules. Common examples include ruthenium complexes like [Ru(bpy)_3] / K{2 + } (bpy = 2,2'-bipyridine), and luminol.

[0126] Co-reactants: These substances participate in the electrochemical reaction to generate the excited state of the luminophore. Tripropylamine (TPrA) is often used as a co-reactant with ruthenium complexes. Hydrogen peroxide (H2O2), reacts with luminol in the presence of HRP to produce a chemiluminescent signal. Hence, ECL may consist of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a substrate on which HRP-conjugated antibodies (or other probes, such as HRP-streptavidin) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. PV ref: 84489PC01

[0127] 15

[0128] More specifically HRP conjugated with an anti-biotin antibody or HRP conjugated with a streptavidin may be bound to the RCA product (containing biotin-labelled nucleotides) and signals are generated by adding ECL. Signals can subsequently be detected as outlined above.

[0129] Colorimetric assay

[0130] In the context of the present invention, a "colorimetric assay" refers to a method wherein a chromogenic substrate (such as TMB (3,3',5,5'-tetramethylbenzidine)) is oxidized by the enzyme horseradish peroxidase (HRP) in the presence of an oxidizing agent, such as hydrogen peroxide (H2O2). This reaction produces a blue colour. The change can be measured e.g. spectrophotometrically, providing a quantitative readout of the assay.

[0131] More specifically HRP conjugated with an anti-biotin antibody or HRP conjugated with a streptavidin is bound to the RCA product (containing biotin-labelled nucleotides) and signals are generated by adding a chromogenic substrate (such as TMB) and an oxidizing agent, such as hydrogen peroxide (H2O2). Signals can subsequently be detected as outlined above.

[0132] Mycobacterium

[0133] Mycobacterium is a genus of over 170 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (M. tuberculosis') and leprosy (M. leprae) in humans. It also includes Mycobacterium smegmatis. The Mycobacterium tuberculosis complex (MTC or MTBC) is a genetically related group of Mycobacterium species that can cause tuberculosis in humans or other animals. It includes Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis and the Bacillus Calmette-Guerin strain, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium dassie, and Mycobacterium mungi.

[0134] Plasmodium

[0135] A genus of parasitic protozoa that causes malaria in humans and other animals. The parasite is most often transmitted through the bite of an infected mosquito. Strains of Plasmodium causing malaria in humans are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. All human malaria-causing Plasmodium species express a life essential PV ref: 84489PC01

[0136] 16 type IB topoisomerase, pTOPl that cleaves and ligate partially double-stranded DNA in a specific manner. Hence, pTOPl can be detected by the assay as it converts a single-stranded sequence that folds upon itself to generate a hairpin structure with a double-stranded stem that is cleaved and ligated specifically by pTOPl.

[0137] Orthopoxyiruses

[0138] Orthopoxvirus is a genus of viruses in the family Poxviridae and subfamily Chordopoxvirinae. Vertebrates, including mammals and humans, and arthropods serve as natural hosts. Diseases associated with this genus include smallpox, cowpox, horsepox, camelpox, and mpox. The most widely known member of the genus is Variola virus, which causes smallpox. Mpox, formerly known as monkeypox, is an infectious viral disease that can occur in humans and other animals. Mpox DNA topoisomerase 1 (mpoxTOPl) is a highly conserved viral DNA cleaving and ligating enzyme with a small size and low homology to human proteins.

[0139] Capture moiety

[0140] In the present context the term "capture moiety" refers to moieties attached to the oligonucleotides or RCA products of the invention. It is to be understood that preferably the different capture moieties (first, second and third) are different types of moieties. Also, in the present context the term "capture moiety" is to be understood as a moiety which can be bound by an antibody and thus be "captured". Examples of capture moieties are biotin, FITC, FAM, DIG (digoxigenin), TAMRA, Cy5, Cy3. The skilled person will be able to identify other suitable capture moieties.

[0141] • A first capture moiety may be incorporated during RCA, such as biotin. Thus, the first capture moiety is used for detecting the RCA product.

[0142] • A second capture moiety may be attached to the 5'-end or the 3'-end of the DNA oligonucleotide (1). As outlined in higher detail below, such second capture moiety at the 5 '- or 3 '-end of the substrate DNA oligonucleotide can be used for removing uncircularized DNA oligonucleotide (1), since the second capture moiety will be removed during circularization of the substrate DNA oligonucleotide. Removal can e.g. take place in a lateral flow device before the RCA products reach the detection line. PV ref: 84489PC01

[0143] 17

[0144] Attachment to the 5'-end is required for assays adapted for detection of type 1A topoisomerases such as mTOPl present in members of the MTBC.

[0145] Attachment to the 3'-end is required for assays adapted for detection of typelB topoisomerases such as pTOPl, and mpoxTOPl present in Plasmodium spp. and mpox, respectively.

[0146] The above difference is due the enzymatic action of the target TOPI enzyme present in the respective organisms.

[0147] • A third capture moiety may be attached to the primer, preferably at the 5'-end. Thus, such third capture moiety can be used for capturing the RCA product, e.g. in a lateral flow device, followed by detection via the first capture moiety.

[0148] Method for determining whether a sample comprises a pathogen or not

[0149] Embodiments of the method of the invention is illustrated in figure 1, showing how circularization can be performed in solution and subsequently RCA can be performed either with the primer coupled to a solid support (Figure 1A) or RCA is performed in solution followed by detection in a lateral flow assay (Figure IB). By comparing Examples 2 and 3 it can be seen that it has surprisingly been found that circularization in solution followed by RCA incorporating labelled nucleotides is an efficient combination. Thus, an aspect of the invention relates to a method for determining whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases, the method comprising a) having a sample, such as a saliva sample or tongue swab, provided; b) providing a single stranded DNA oligonucleotide (1) comprising:

[0150] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl) (2);

[0151] - a primer-annealing element, complementary to a DNA oligonucleotide (4); and PV ref: 84489PC01

[0152] 18 c) incubating in solution, preferably in the presence of Mg2+ions, the biological sample of step a) with the oligonucleotide (1) from step b), wherein if the DNA modifying enzyme, such as mTOPl (2) is present in the sample from step a), the single stranded DNA oligonucleotide of step b) is circularized (3); d) hybridizing the oligonucleotide (1) from step c) to the DNA oligonucleotide (4); e) determining the level of circularized oligonucleotides in the sample from step c) using Rolling Circle Amplification (RCA) with incorporation of labelled nucleotides, preferably the label is a first capture moiety, such a biotin moiety, such as biotin-dCTP; f) comparing said determined level to a reference level; and g) determining that if said level is above said reference level it is indicative of the sample comprising the pathogen; or if said reference level is equal to or below said reference, it is indicative of the sample not comprising the pathogen.

[0153] In an embodiment, the pathogen is selected from the group consisting of Mycobacterium spp., Plasmodium spp., and poxviruses, such as smallpox, cowpox, horsepox, camelpox, and mpox, Leishmania spp., and trypanosoma spp.. As shown in the example section, the method of the invention has been confirmed for a number of pathogens expressing the relevant DNA modifying enzymes. Example 3 shows detection of mTopl. Examples 9-10 show that Plasmodium Topoisomerase 1 (pTOPl) can be detected using the assay of the invention, including detection in saliva from confirmed malaria-positive individuals. Example 11 shows that the assay of the invention allows for detection of mpox (mpoxTOPl) in saliva.

[0154] In a preferred embodiment, the Mycobacterium spp. is from the Mycobacterium tuberculosis complex, preferably the human Mycobacterium tuberculosis complex.

[0155] The pathogen may also be a pathogen often found in e.g. food or feed. Thus, in an embodiment, the pathogen is selected from the group consisting of Listeria spp., E. coll, Pseudomonas spp., Campylobacter spp., and Salmonella spp. PV ref: 84489PC01

[0156] 19

[0157] Step a )

[0158] Different types of samples may be used in the method of the invention. Thus, in an embodiment, the sample is a biological sample, such as selected from the group consisting of saliva, tongue swab, sputum, blood plasma, blood serum, urine, cell smear, faeces, cerebrospinal fluid, and a biopsy, preferably being a saliva sample or tongue swab, more preferably a lysed saliva sample or tongue swab.

[0159] In a preferred embodiment, the sample is a saliva sample or tongue swab.

[0160] In another embodiment, the sample is selected from the group consisting of a food sample, a water sample, an environmental sample, such as liquid, water, soil, air, and plant samples, such as seeds or said sample is selected from the group consisting of animal feed or food for human consumption, such as meat and meat products, dairy products, such as milk, cream, fermented milk products, cheeses and butter, plants, such as vegetables and fruit, fish and fish products, egg and egg products, condiments and spices.

[0161] In an embodiment, the sample is lysed, preferably lysis is performed by i) freeze and thawing the sample, preferably followed by vortexing with a solid material, such as glass beads; ii) bead beating; and / or iii) using lytic mycobacteriophages.

[0162] In yet an embodiment, the subject is a mammal, preferably a human.

[0163] Step b

[0164] Certain types of DNA modifying enzymes, such as topoisomerases, have preference for certain binding motifs. Thus, in an embodiment, the binding element of the single stranded oligonucleotides (1) of step b) is selected from the groups consisting of:

[0165] • 5'-CGCTTG-3', 5'-CGCTTC-3', 5'-CTCTTG-3', and 5'-CTCTTC-3' (mTOPl); or

[0166] • CCCTT (mPoxTOPl) pTOPl has no preferred base sequence for binding, cleavage and ligation. It can be distinguished from other enzymes by using a DNA oligonucleotide that folds into a hairpin structure with a protruding 5'-end and without a sequence different from the sequence CCCTT at the end. pTOPl can cleave close to the 3 '-end of such oligonucleotide and form a circle by ligating the protruding 5'-end. PV ref: 84489PC01

[0167] 20

[0168] In an embodiment, the binding element of the single stranded oligonucleotides (1) of step b) is located within the first 50 nucleotides, preferably within the first 40 nucleotides, more preferably within the first 30 nucleotides, most preferably within the first 20 nucleotides of the 5'-end.

[0169] In yet an embodiment, the sequence element of the oligonucleotide (1) 5' to primer-annealing element, has

[0170] • a percentage of nucleotides complementary to the labelled nucleotides to be incorporated in step e) in the range 0-10%, such as 0-5%, such as 0- 2% preferably 0%; and / or

[0171] • a number of nucleotides complementary to the labelled nucleotides to be incorporated in step e) in the range 0-5, such as 0-3, such as 0-2, preferably 0-1 and or more preferably 0.

[0172] An advantage of such a setup is that background noise due to extension on uncircularized oligonucleotides (1) is reduced since the number of incorporated labelled nucleotides is minimized.

[0173] In an embodiment, the sequence element of oligonucleotide (1) 5' to the primerannealing element, has a length in the range 5-30 nucleotides, such as 10-20 nucleotides.

[0174] In yet an embodiment, the primer-annealing element of the single stranded oligonucleotides (1) of step b) is complementary to a DNA oligonucleotide (4) attached to a solid support (5), such as a glass slide.

[0175] In a further embodiment, the oligonucleotide (4) attached to a solid support (5) is modified with an amine, preferably at the 5'-end.

[0176] In an embodiment, the DNA oligonucleotide (4) comprises a third capture moiety, such as a FAM moiety or FITC moiety, preferably the third capture moiety is at the 5'-end. Preferably the third capture moiety is different from the first capture moiety and the second capture moiety. It should be recalled that a FITC antibody also binds to FAM. By having a label at the 5'-end of the DNA oligonucleotide (4) is that it can still work as a primer while still being able to be captured by an antibody, preferably in a lateral flow device setting. Thus, in a preferred PV ref: 84489PC01

[0177] 21 embodiment, the DNA oligonucleotide (4) is in solution and / or not being bound to a solid support.

[0178] In an embodiment, the primer annealing element of the single stranded oligonucleotide (1) of step b) is at least 25 nucleotides long, preferable at least 20 nucleotides long, more preferably at least 18 nucleotides long, such as having a length in the range 18-25 nucleotides.

[0179] In yet an embodiment, the single stranded DNA oligonucleotide (1) of step b) has a length in the range 80-120 nucleotides, such as 90-110, preferably in the range 95-105 nucleotides, such as 98-102.

[0180] Different DNA oligonucleotides (1) have been designed as outlined in the sequence list. These are optimized for circularization by different DNA modifying enzymes. Thus, in an embodiment, the single stranded oligonucleotide (1) of step b) is selected from the group consisting of SEQ ID NO': 1-12.

[0181] In a further embodiment, the oligonucleotide (1) comprises a second capture moiety at the 5'-end or the 3'-end, said second capture moiety preferably being different from the first capture moiety and the third capture moiety, such as the second capture moiety being FAM or FITC. As outlined in example 16, such a setup may be used to improve the signal to noise ratio in a lateral flow setting.

[0182] In yet an embodiment, the second capture moiety is positioned in the first oligonucleotide allowing it to be cleaved of after circularization of the oligonucleotide (1) by enzymatic action of the DNA modifying enzyme. It should be recalled that the substrate oligonucleotides (1) are designed so that a short fragment of the oligonucleotide is cleaved of during the cleavage and ligation reaction (see e.g. WO2008 / 148392 Al).

[0183] Step c

[0184] In an embodiment, an exonuclease treatment step is included after step c) and preferably before step d), preferably the exonucleases are inactivated, preferably by heat-inactivation, before hybridization of the DNA oligonucleotide (1) to the PV ref: 84489PC01

[0185] 22

[0186] DNA oligonucleotide (4). Exonuclease treatment may improve the signal-to-noise ratio, by removing uncircularized DNA oligonucleotides (1).

[0187] The buffer used in step c) may also be optimized. Thus, in an embodiment, step c) is performed with a concentration of MnCh below 1 mM, such as below 0.1 mM MnCh, preferably below 0.01 mM MnCh, and more preferably substantially in the absence of MnCh. or in the absence of MnCh.

[0188] In another embodiment, in step c) Mg2+ions are present in the range 2-50 mM, preferably in the range 10-40 mM, more preferably in the range 20-40 mM, or in the range 25-35 mM.

[0189] In yet another embodiment, in step c) Na+ions are present in an amount of 50- 3000 mM, such as at a concentration in the range 100-2000 mM, preferably 200- 1000 mM, more preferably 300-500 mM (may be relevant for detection of M. smegmatis) or wherein in step c) Na+ions are present in an amount of 0-3000 mM, such as at a concentration in the range 0-2000 mM, preferably 0-1000 mM, more preferably 0- 100 mM or 10-100 mM or 10-50 mM.

[0190] In a further embodiment, in step c) the solution contains

[0191] • below 0.1 mM Mn2+, preferably below 0.01 mM Mn2+;

[0192] • in the range 10-40 mM Mg2+, preferably in the range 20-40 mM Mg2+; and

[0193] • in the range 200-1000 mM Na+; or contains

[0194] • below 0.1 mM Mn2+, preferably below 0.01 mM Mn2+;

[0195] • in the range 10-40 mM Mg2+, preferably in the range 20-40 mM Mg2+; and

[0196] • in the range 0-100 mM Na+or 10-100 mM Na+.

[0197] In examples 6 and 17 data is provided showing optimization of the buffer conditions.

[0198] Step e

[0199] Different types of labelled nucleotides may be used in step e) of the method of the invention, as long as they can subsequently be detected, such as by an antibody. PV ref: 84489PC01

[0200] 23

[0201] Thus, in an embodiment, in step e) biotin-labelled nucleotides are used, such as biotin-dCTP or fluorescently labelled nucleotides, or labelled nucleotides recognizable by an antibody.

[0202] In an embodiment, in step e) the polymerase phi29 is used for RCA.

[0203] In another embodiment, the level of circularized oligonucleotides in the sample in step e), is determined by an assay selected from a chemiluminescence assay and a colorimetric assay.

[0204] In yet an embodiment, the level of circularized oligonucleotides in the sample in step e), is determined by chemiluminescence assay, wherein HRP conjugated with an anti-biotin antibody and / or HRP conjugated with a streptavidin is bound to the RCA product, containing biotin-labelled nucleotides, and signals are generated by adding ECL and an oxidizing agent, such as hydrogen peroxide (H2O2), followed by detection.

[0205] In yet a further embodiment, the level of circularized oligonucleotides in the sample in step e), is determined by a colorimetric assay, wherein HRP conjugated with an anti-biotin antibody or HRP conjugated with streptavidin is bound to the RCA product, containing biotin-labelled nucleotides, and signals are generated by adding a chromogenic substrate, such as TMB (3,3',5,5'-tetramethylbenzidine), and an oxidizing agent, such as hydrogen peroxide (H2O2), followed by detection.

[0206] In an embodiment, the chromogenic substrate is selected from the group consisting of TMB, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine dihydrochloride), DAB (3,3'-diaminobenzidine), and BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium), preferably being TMB.

[0207] In a preferred embodiment, the level of RCA product is determined in a lateral flow assay. As outlined in the example section it has surprisingly been found that a single label on the primer is sufficient for capturing each RCA product in the lateral flow assay. PV ref: 84489PC01

[0208] 24

[0209] In an embodiment, the lateral flow device comprises a membrane material comprising nitrocellulose, such as CN95, CN140 or CN180DX.

[0210] In yet an embodiment, the lateral flow assay comprises a capture element adapted for capturing uncircularized oligonucleotides (1) that can act as a template for the polymerase, such as through a capture moiety present on the 5'- end or the 3'end of the oligonucleotides (1), preferably being adapted to capture the uncircularized oligonucleotides (1) that acts as a template for primer elongation by the polymerase before they reach the test line in the lateral flow assay, preferably the test line contains anti-DIG antibodies or anti-biotin antibodies. As outlined in example 16, such a setup may improve the signal to noise ratio.

[0211] In an embodiment, the RCA step also includes the addition of a branching oligonucleotide, such as SEQ ID NO: 17. The skilled person will be able to design alternative branching oligos by knowing the sequence of the exact generated RCA product. Rolling Circle Amplification (RCA) branching is a technique that modifies the standard linear RCA to create multiple branches of amplified DNA, increasing the amplification yield and sensitivity. This is achieved by introducing branching primers that, along with the primary strand displacement activity of the (phi29) DNA polymerase, lead to a more complex, branched DNA structure with exponential signal amplification. This enhanced product generation is beneficial for applications requiring high sensitivity, such as the detection of low copy numbers of DNA and / or to increase signal-to-noise ratio. In example 19 branching is tested by the addition of a branching oligo.

[0212] Step f

[0213] In an embodiment, the reference level of step f) is determined in a subject or a group of subjects without a corresponding pathogenic infection, such as a Mycobacterium tuberculosis infection.

[0214] In another embodiment, the reference level of step f) is determined using the same method as used to determine the level of circularized DNA in step e).

[0215] In a preferred embodiment, PV ref: 84489PC01

[0216] 25 if said level is above said reference level it is indicative of said subject being infected with the pathogen, such as with Mycobacterium spp; or if said reference level is equal to or below said reference, it is indicative of said subject not being infected with the pathogen, such as with Mycobacterium spp.

[0217] Kit of parts

[0218] The oligonucleotides and the lateral flow device of the invention may form part of a kit of parts. Thus, an aspect of the invention relates to a kit of parts comprising a) a single stranded DNA oligonucleotide (1) comprising:

[0219] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl) (2);

[0220] - a primer-annealing element, complementary to a DNA oligonucleotide (4); b) a labelled DNA oligonucleotide (4), suitable for acting as a primer for an RCA reaction of a circularized version of the single stranded DNA oligonucleotide (1); and c) a lateral flow device adapted to detect an RCA product comprising labelled nucleotides, such as biotins; d) optionally reaction components, such as a lysis buffer, exonuclease mix, RCA mix and / or detection solutions; e) optionally, a gel filtration column, adapted for removing excess reagents; and f) optionally a branching oligo, such as SEQ ID NO: 17.

[0221] In an embodiment, the lateral flow device is adapted to detect an RCA product comprising a capture moiety adapted for capturing uncircularized oligonucleotides (1) that act as template for primer elongation by the polymerase, such as through a capture moiety present on the 5'-end or the 3'-end of the oligonucleotides (1), preferably being adapted to capture the uncircularized oligonucleotides (1) that template primer elongation by the polymerase before they reach the test line in the lateral flow assay, preferably the test line contains anti-DIG antibodies or antibiotin antibodies. PV ref: 84489PC01

[0222] 26

[0223] Uses

[0224] The kit of part of the invention may have certain uses. Thus, a further aspect of the invention relates to the use of the kit of parts according to the invention for determining, whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases.

[0225] In an embodiment, the use is for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with Mycobacterium spp, preferably the Mycobacterium tuberculosis complex.

[0226] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. For example, the embodiments described under the method of the invention may equally apply to the "kit of part" aspect and the "use" aspect of the invention and vice versa.

[0227] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0228] The invention will now be described in further details in the following non-limiting examples.

[0229] Examples

[0230] Example 1. The method of the invention

[0231] In SOLEAD, linear DNA oligonucleotides (1) are turned into DNA circles (3) by Mycobacteria spp. Topoisomerasel (mTOPl) (2) (or other target enzymes e.g. Plasmodium Topoisomerase 1 (pTOPl) or mpox Topoisomerase 1 (mpoxTOPl)) in solution, whereafter the circles are hybridized to a primer (via a DNA element (4) complementary to the primer) to support RCA, where incorporation of biotinylated nucleotides occurs. After RCA, the products can be detected on a solid support where HRP conjugated with an anti-biotin antibody or streptavidin is bound to the PV ref: 84489PC01

[0232] 27

[0233] RCA product, and signals are generated by adding ECL (for chemiluminescence readout) or TMB (for colorimetric readout) (Figure 1A). This readout surprisingly only works if the circles are made in solution, which could not be predicted.

[0234] As an alternative readout, the biotinylated RCA products generated in solution by using an e.g. 5 '-FAM labelled primer can also be applied to a lateral flow membrane functionalized with an anti-FITC antibody that captures the RCA products at a test line. The RCA products are subsequently labelled with antibiotin antibody-conjugated gold-nano particles resulting in a red-colored testline (Figure IB). It was not expected that the RCA products could efficiently flow through the membrane. It was also surprising that the RCA product could be caught at the test line based on a single FAM molecule (coupled to the primer), since there was a risk that the FAM molecule would be hidden in the bulky structure of the RCA product.

[0235] Experimental proof of concept and validation has been achieved, as illustrated examples 2 to 17.

[0236] Example 2 - ECL readout on solid support setup

[0237] Aim of study

[0238] The previous readout for the detection of mTOPl is based on fluorescent labelling of the RCA products followed by microscopy. Franch et al. Nanoscale, 2019,11, 587-597. Here it is investigated if the readout can be modified for detection using an ECL-based readout.

[0239] Materials and Methods

[0240] A HD slide (Surmodics # DHD1 - TRIDIA™ HD Activated Slides) was cut into an appropriate size. The slide was attached to custom-made silicone grid to create delimited rectangular-shaped wells termed the Wellmaker. 10 pM of the 5'-Amine ID33 primer SEQ ID NO: 14 in 300 mM Na3PO4 pH 8 was coupled to the wells. The Wellmaker was incubated overnight in a humidity chamber with saturated NaCI. Subsequently, it was blocked in a buffer containing 50 mM Tris, 50 mM Tris- HCI, and 50 mM ethanolamine, pH 9) for 30 min at 50 °C and washed twice in ddH2O before washing in a buffer containing 4x SSC, and 0.1% SDS for 30 min at 50 °C and finally washed twice in ddH2O.

[0241] 10 pmol of the mTOPl specific SEQ ID NO: 1 was hybridized onto the wells of the Wellmaker in a buffer containing 10 mM tris-HCI pH 7.5, ImM EDTA and 400 mM NaCI for 1 hour at 37°C in a humidity chamber. The Wellmaker was then washed PV ref: 84489PC01

[0242] 28 for 1 minute in 0.1 M Tris-HCI pH 7.5, 150 mM NaCI, 0.3% SDS, 1 minute in 0.1 M Tris-HCI pH 7.5, 150 mM NaCI, 0.05% Tween 20, and 1 minute in 70% ethanol. Purified mTOPl (doi: 10.1039 / c8nr07850e) mediated cleavage and ligation of the DNA substrate molecule was initiated by addition of a buffer containing 10 mM Tris-HCI (pH 7.5), 200 mM NaCI, lOmM MgCh, ImM DTT and 10 mM MnCh with or without purified mTOPl. The Wellmaker was incubated for 1.5 hours at 37 °C in a humidity chamber. After incubation, the Wellmaker was washed for 1 minute in 0.1 M Tris-HCI (pH 7.5), 150 mM NaCI, 0.3% SDS, 1 minute in 0.1 M Tris-HCI (pH 7.5), 150 mM NaCI, 0.05% Tween 20, and 1 minute in 70% ethanol.

[0243] Rolling circle amplification (RCA) was performed in lx Phi29 buffer (50 mM Tris- HCI pH 7.5, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT) supplemented with 0.2 pg BSA, 100 pM dATP, 100 pM dTTP, 100 pM dGTP, 90 pM dCTP, 10 pM biotin- dCTP, and 1 Unit Phi29 polymerase using SEQ ID NO: 14 as the primer. The reaction was carried out for 2 h in a humidity chamber at 37 °C. The Wellmaker was then washed for 1 minute in 0.1 M Tris-HCI (pH 7.5), 150 mM NaCI, 0.3% SDS, 1 minute in 0.1 M Tris-HCI (pH 7.5), 150 mM NaCI, 0.05% Tween 20, and 1 minute in 70% ethanol. Subsequently, HRP-conjugated anti-biotin antibody was diluted 1 :300 in a buffer containing lx TBST (20 mM Tris-HCI pH 9, 150 mM NaCI, 0.05% Tween20 pH 9) supplemented with 5% skimmed dry milk and 5% BSA and added to the wells of the Wellmaker for 1 h at room temperature. The Wellmaker was washed 3 x 5 min in lx TBST and finally 2 pL of 1 : 1 ECL mixture (Cytiva) was added to allow chemiluminescence readout using a CCD camera.

[0244] Result

[0245] The result shown in Figure 2 demonstrates that a higher chemiluminescent signal in samples where mTOPl was not added compared to samples incubated with added mTOPl was obtained. This was the opposite of the expected result. Conclusion

[0246] The solid support assay for detection mTOPl could not be adapted to an ECL based readout based on the coupling of anti-biotin-antibody HRP to biotinylated RCA products.

[0247] Example 3 - mTOPl detection by ECL-based readout using new DNA substrate sequences and circularization in solution

[0248] Aim of study PV ref: 84489PC01

[0249] 29

[0250] The result shown in example 2, was surprising since we did not observe similar results with DNA substrates for other enzymes e.g. human TOPI.

[0251] The hypothesis was, that the signal obtained when no mTOPl was added to the reaction, was a consequence of the incorporation of biotinylated nucleotides in the product arising when phi-29 used the un-circularized substrate as a template.

[0252] To minimize the potential incorporation of the utilized biotin functionalized dCTPs during the amplification of non-circularized substrates, we designed new substrates with minimum numbers of the base "G" 5 'to the primer annealing site. The substrates were tested out in an assay setup where circles were made in solution before, they were hybridized to surface-attached primers.

[0253] Method

[0254] The Wellmaker was prepared as described in example 2 except that the DNA substrate sequences were not hybridized to the surface-anchored primer but directly incubated with mTOPl in solution. 10 pmol of the oligonucleotide sequences (SEQ ID Nos: 2, 3, 4, 5, 6) were incubated in the presence or absence of purified mTOPl in a buffer containing 10 mM Tris-HCI (pH 7.5), 200 mM NaCI, lOmM MgCh, ImM DTT, and lOmM MnC for 1.5 hours at 37 °C in a humidity chamber.

[0255] The obtained circles were hybridized to the wells for 1 hour at 37 °C.

[0256] Subsequently the Wellmaker was washed and RCA performed with biotinylated nucleotides as described in example 2.

[0257] Results

[0258] The example includes investigations of SEQ ID NOs: 2, 3, 4, 5, and 6.

[0259] In each case, incubation of the substrates with SEQ ID NO: 2, 3, 4, 5, or 6 with purified mTOPl resulted in a higher chemiluminescent signal than the negative sample incubated without mTOPl as shown in Figure 3.

[0260] Conclusion

[0261] The SOLEAD assay worked with an ECL-based readout when using several of the new sequences including SEQ ID NOs: 2, 3, 4, 5, and 6. It was unexpected that the incubation with the substrate in solution would make a difference.

[0262] Example 4 - The effect of exonuclease digestion for the SOLEAD using the ECL based readout

[0263] Aim of study PV ref: 84489PC01

[0264] 30

[0265] We aimed to investigate the performance of the original substrate (SEQ ID NO: 1) compared to the best of the new sequences (SEQ ID NO: 6), and we wanted to test the hypothesis that amplification of the unreacted substrate potentially resulted in the background signal observed in example 2.

[0266] The performance of the new substrates and protocol were tested using extracts from the model organism M. smegmatis. We tested SEQ ID NO: 6 and the original SEQ ID NO: 1 and tested the effect of adding exonuclease (to remove undigested substrate) to the circle mixture before RCA. This was possible with the new protocol since circles were made in solution before they were hybridized with the primers.

[0267] Methods

[0268] The Wellmaker was prepared as described in example 3. M. smegmatis mc2155 cultures were grown in growth media (7H9 liquid medium supplemented with carbenicillin (50 mg ml-1), cycloheximide (10 mg ml-1), 10% ADC, 1 mM CaCh and 0.25 vol% of TWEEN-80) to OD600 = 0.6 giving a 2xl06CFU / mg as previously determined (doi: 10.1039 / c8nr07850e). Pellets of lOmg of M. smegmatis were resuspended in 100 pl of saliva (collected in Denmark) supplemented with of lx Proteases inhibitors (Roche, Merck#04693159001). The resuspended pellets were frozen for 2 minutes on dry-ice, thawed in hands for two times followed by vortexing for 3 times for two minutes using the Vortexer genie (Scientific Industries #SI-D258). The obtained extracts from 40000CFU / pl of M. smegmatis spiked-in saliva were incubated with 40 pmol of DNA oligonucleotide SEQ ID NO: 1 or SEQ ID NO: 6 in a buffer containing 10 mM Tris-HCI (pH 7.5), 200 mM NaCI, lOmM MgCh, ImM DTT and lOmM MnCh for 1.5 hours at 37 °C. Extract from saliva alone was used as negative control. The obtained circles were subjected to incubation in absence or presence of 8U of Exonuclease I and 80U of exonuclease III in a buffer containing 10 mM Bis-Tris-Propane-HCI, 10 mM MgC , 1 mM DTT (pH 7 @ 25°C) for 1 hour at 37 °C.

[0269] The digested circles were hybridized to the wells of the Wellmaker, blocked and washed as described in example 2, for 1 hour at 37 °C. Subsequently the Wellmaker was washed and RCA performed with biotinylated nucleotides as described in example 2.

[0270] Result

[0271] We found that for SEQ ID NO: 1 it is necessary to add an exonuclease digestion step to the protocol to see signals above the background when testing M. PV ref: 84489PC01

[0272] 31 smegmatis extract, while exonuclease treatment makes little difference for SEQ ID NO: 6 in terms of signal-to-noise (figure 4).

[0273] Conclusion

[0274] Exonuclease treatment of circle mixture before RCA improves the readout for some sequences and not for others.

[0275] Example 5 - The effect of DNA substrate concentration.

[0276] Aim of study

[0277] To identify the optimal DNA substrate concentration for the SOLEAD assay.

[0278] Methods

[0279] The preparation of the Wellmaker and the extraction of M. smegmatis spiked-in saliva were performed as described in example 4. The obtained extracts from 40000CFU / pl of M. smegmatis spiked-in saliva were incubated with 10, 20, 40, or 80 pmol of DNA oligonucleotide sequence 1 in a buffer containing 10 mM Tris-HCI (pH 7.5), 200 mM NaCI, lOmM MgCh, ImM DTT and lOmM MnCh for 1.5 hours at 37 °C and digested with exonucleases as described in example 4. The RCA was performed was performed in lx Phi29 buffer (50 mM Tris-HCI pH 7.5, 10 mM MgCh, 10 mM (NH4)2SC>4, 4 mM DTT) supplemented with 0.2 pg BSA, 1 mM dNTP, and 1 unit of Phi29 polymerase. The reaction was carried out for 1 h at 37 °C in a humidity chamber and subsequently washed as previously described. The rolling circle products were detected by hybridizing 2 pM of the fluorescent probe SEQ ID NO: 16 in a buffer containing 2x SSC, 20% formamide, and 5% glycerol for 30 min in a humidity chamber at 37 °C. The slides were washed as previously described, mounted with Vectashield, and covered with cover glass. The slides were analyzed in an Olympus 1X73 fluorescence microscope with a 60x objective. Twelve images of the fluorescent signals were acquired for each sample and quantified using Image J.

[0280] Results

[0281] It was found that 40 pmol of substrate SEQ ID NO: 1 enhanced the signal without creating an increased background (Figure 5).

[0282] Conclusion

[0283] 40 pmol of DNA substrate is optimal concerning signal-to-noise. PV ref: 84489PC01

[0284] 32

[0285] Example 6 - Optimal buffer conditions for the circularization reaction in the SOLEAD assay

[0286] Aim of study

[0287] To identify optimal conditions for generating DNA circles, the optimal conditions for circularization by extracts from the model organism M. smegmatis spiked in saliva were investigated concerning NaCI and the presence of divalent cations. Method

[0288] The preparation of the Wellmaker and the extraction of M. smegmatis spiked-in saliva were performed as described in example 4. The obtained extracts from 40000CFU / pl of M. smegmatis spiked-in saliva were incubated with 10 pmol of oligonucleotide sequence 1 in the presence of in buffers containing 10 mM Tris- HCI (pH 7.5), 200 mM NaCI, lOmM MgCh, ImM DTT and lOmM MnCh or 10 mM Tris-HCI (pH 7.5), 200 mM NaCI, 30mM MgCh or 10 mM Tris-HCI (pH 7.5), 400 mM NaCI, 30mM MgCh or 10 mM Tris-HCI (pH 7.5), 800 mM NaCI, 30mM MgCh or 10 mM Tris-HCI (pH 7.5), 1500 mM NaCI, 30mM MgCh for 1.5 hours at 37 °C.

[0289] RCA and microscope visualization was carried out as described in example 5.

[0290] Result

[0291] As evident from figure 6, we found that the optimal signal was achieved when using 400 mM NaCI and 30 mM MgCh and no MnCh using SEQ ID NO: 1.

[0292] Conclusion

[0293] Our data demonstrate that increasing the concentration of MgCh and omitting MnCh in the reaction buffer gives an increased signal. This is in contrast to all published protocols we have been able to find.

[0294] Example 7 - Detection limit and quantitative detection by SOLEAD with ECL readout

[0295] Aim of study

[0296] To determine if SOLEAD allows for quantitative detection of mycobacteria and to identify the detection limit, extracts made by a dilution series of the model organism M. smegmatis were analyzed.

[0297] Method

[0298] The preparation of the Wellmaker and the extraction of M. smegmatis spiked-in saliva were performed as described in example 4. The extract was diluted in saliva to 20000 or 10000 CFU / ul. Extract of E.coli (strain RY13), grown in LB medium at a concentration of 40000 CFU / ul was also included. The extracts were incubated PV ref: 84489PC01

[0299] 33 with 40 pmol of oligonucleotide SEQ ID NO: 1 in a buffer containing 10 mM Tris- HCI (pH 7.5), 400 mM NaCI, 30mM MgCh, ImM DTT for 1.5 hours at 37 °C and analyzed as described in example 4.

[0300] Result

[0301] Analyzing decreasing concentration of M. smegmatis resulted in decreasing signals with a detection limit of 1000 CFU / |JL as shown in in Figure 7.

[0302] Conclusion

[0303] SOLEAD allows for quantitative detection of M. smegmatis with a detection limit of 1000 CFU / IJL.

[0304] Example 8 - Detection of Mtb (mycobacteria from the tuberculosis complex) in saliva or sputum from TB-positive individuals.

[0305] Aim of study

[0306] We wanted to investigate if the SOLEAD assay can detect the presence of the tuberculosis complex (MTb) in saliva from individuals with confirmed TB and compare the results to those obtained when testing sputum, which is the standard specimen for TB diagnosis.

[0307] Method

[0308] The preparation of the Wellmaker and the extraction of saliva or sputum from infected individuals (collected in Guinea Bissau) were performed as described in example 4 with the use of SEQ ID NO: 1. Negative saliva samples, collected in Denmark, were included. The extract was diluted in saliva to 20000 or 10000 CFU / ul. Extract of f. coli (strain RY13), grown in LB medium at a concentration of 40000 CFU / ul was also included. The extracts were incubated with 40 pmol of oligonucleotide sequence 1 in a buffer containing 10 mM Tris-HCI (pH 7.5), 400 mM NaCI, 30mM MgC , ImM DTT, for 1.5 hours at 37 °C and analyzed as described in example 4.

[0309] Result

[0310] Analysis of saliva and sputum from individuals with confirmed TB resulted in signals above the background signal obtained when analyzing saliva from a TB- negative individuals. Saliva and sputum resulted in signals with similar intensity, which was surprising as sputum is the standard specimen, and qPCR protocols such as GeneXpert performs less optimal with saliva compared to sputum (Figure 8).

[0311] Conclusion PV ref: 84489PC01

[0312] 34

[0313] SOLEAD allows for detection of Mtb in saliva and in sputum from TB positive individuals. This is the first example of detection of TB in clinical samples with SOLEAD and to our knowledge the first example of detection of TB in saliva and sputum with equal intensity.

[0314] Example 9 - Detection of Plasmodium Topoisomerase 1 (pTOPl) using the SOLEAD protocol

[0315] Aim of study

[0316] The aim of this study is to investigate if the SOLEAD protocol can also be used to detect other target enzymes such as Plasmodium Topoisomerase 1 (pTOPl). An aim was also to detect the influence of exonuclease treatment. Thus, instead of one of the sequences SEQ ID Nos: 1-6 (specific for mTOPl) we used SEQ ID NO: 7, which is specific for pTOPl.

[0317] 12 biotinylated dCTPs can be incorporated in an amplification product when using uncircularized SEQ ID NO: 7 as a template. This can lead to binding of HRP and result in high background signal as is the case for SEQ ID NO: 1. We, therefore, investigated if exonuclease treatment was necessary to allow for the ECL-based readout of SOLEAD specific for pTOPl.

[0318] Methods

[0319] The circularization was carried out by incubating 1 ng / pL pTOPl with 5 pmol of the oligonucleotide SEQ ID NO: 7 in the presence of a buffer containing 10 mM Tris-HCI pH 7.5, and 5 mM EDTA and supplemented with 250 mM NaCI for 1 h at 37 °C. The circles were subjected to digestion and analyzed as described in example 4.

[0320] Results

[0321] Incubation of SEQ ID NO: 7 with pTOPl resulted in signals above background and exonuclease digestion did not affect the result (Figure 9).

[0322] Conclusion

[0323] The result demonstrated that exonuclease digest was not necessary for the ECL readout when using substrate SEQ ID NO: 7 circularized by Plasmodium Topoisomerase 1 (pTOPl). This result is surprising as 12 biotinylated dCTPs can be incorporated in an amplification product templated by uncircularized substrate. Without being bound by theory, it is believed that other sequences that can be circularized by pTOPl will reduce the background from unreacted substrate by PV ref: 84489PC01

[0324] 35 preventing incorporation of biotinylated dCTPs. Such sequences are presented as SEQ ID NO's: 8 and 9.

[0325] Example 10 - Detection of Plasmodium parasites in saliva from individuals with confirmed malaria using the SOLEAD protocol

[0326] Aim of study

[0327] We wanted to investigate if the SOLEAD protocol with SEQ ID NO: 7, could be used to detect the presence of Plasmodium parasites in saliva from malariapositive individuals. First sample preparation concerning vortex was optimized and thereafter saliva from malaria-negative or positive individuals was tested (n = 61). Methods

[0328] Saliva samples from patients diagnosed with malaria were obtained at the CERMEL, Albert Schweitzer Hospital, Lambarene, Gabon. The patients were diagnosed first using a rapid diagnostic test (RDT) in matching blood, then followed with thick smear microscopy. The study was conducted in accordance with the Declaration of Helsinki, and samples were collected and analyzed following local regulations and guidelines. Patients' samples were obtained from the malaria screening activities conducted at CERMEL according to ethical clearance granted for ongoing studies and informed consent provided by each individual or their representatives. Thick smear microscopy was performed by two independent readers using the Lambarene method (doi: 10.4269 / ajtmh.2001.65.599; doi: 10.1186 / 1475-2875-13-234). A third reading was performed if required (e. g. if the first and second readings differed more than 50% of parasite count or if the same slide was read positively / negatively by different readers). The number of counted parasites per pL blood was recorded. The final parasitemia was determined as the average of the first and second reading (or, if the third reading was performed: the average of the two closest results). In the case of testing by RDTs, the "Paracheck Pf" test was used.

[0329] Saliva from infected and non-infected individuals were analyzed as described for saliva samples infected with tuberculosis (as described in example 4) with the exception of the extraction vortexing time. For the SOLEAD protocol for malaria samples extraction by vortexing for 2x30 seconds, 3x 30 seconds, 4x30 seconds and 5x30 seconds was tested and 5x30 seconds was selected as optimal. Following extraction, the samples were analysed in the SOLEAD protocol by chemiluminescence readout as described in example 4. PV ref: 84489PC01

[0330] 36

[0331] Results

[0332] Incubation of SEQ ID NO: 7 with extracts of saliva from a malaria-positive or a malaria-negative individual prepared by repeated vortexing was tested. The result shows that vortexing five times for 30 seconds gave the best result. Using the sample preparation protocol combined with the SOLEAD assay we successfully tested saliva from a population of 30 malaria-positive and 31 malaria-negative individuals with only a few false positive or false negative readings (figure 10).

[0333] Conclusion

[0334] The result demonstrated the detection of Plasmodium infection in saliva from confirmed malaria-positive individuals using the SOLEAD protocol combined with a simple sample preparation procedure based on vortexing. This was unexpected as previous protocols failed to detect Plasmodium parasites in clinical samples without using droplet microfluidics for sample extractions and enhanced reaction kinetics (DOI: 10.1038 / s41598-018-22378-7).

[0335] Example 11 - Detection of mpox topoisomerase 1 (mpoxTOPl) spiked in saliva

[0336] Aim of study

[0337] To investigate if the SOLEAD protocol could be used in more general terms to detect pathogens in saliva we also investigated if we could measure mpox topoisomerase 1 (mpoxTOPl) spiked in saliva using SEQ ID NO: 10 specific for this enzyme.

[0338] Method

[0339] Serial dilutions of purified mpoxTOPl (https: / / doi.org / 10.3390 / phl6050657) was incubated with 5pmol of mpoxTOPl specific SEQ ID NO: 10: in the presence of saliva. The circularization, RCA and visualization was performed as described in example 4.

[0340] Result

[0341] Decreasing concentrations of mpoxTOPl spiked in saliva gave decreasing signal all above the background signal arising from saliva without mpoxTOPl. This demonstrates that the SOLEAD protocol with SEQ ID NO: 10 can detect mpoxTOPl as a biomarker for mpox (figure 11).

[0342] Conclusion

[0343] The SOLEAD protocol can be used for the detection of an array of human pathogens including mpox. PV ref: 84489PC01

[0344] 37

[0345] Example 12 - Summary of the Lateral Flow Test Strip Reagent Interactions

[0346] As an alternative and user-friendly readout to the ECL-based readout described in example 1 (figure 1A) we tested a lateral flow format as also described in example 1 (figure IB).

[0347] The lateral flow test strip uses a visual detection label e.g. gold colloid which is conjugated to an antibody which binds the analyte of interest. In this case a mouse anti-biotin antibody is used as the gold-conjugated detection antibody (detection label conjugate).

[0348] The detection label conjugate is dried onto a conjugate pad which is fixed at one end of a material such as nitrocellulose membrane. A second analyte specific material, in this case an anti-FITC antibody, is dried onto the nitrocellulose membrane.

[0349] The liquid sample to be analyzed, in this case a rolling circle product (RCP) incorporating biotin and FAM, is placed on to the pad resulting in the resuspension of the detection label conjugate into the sample and allowing any target analyte in the liquid sample to bind to the analyte binding material attached to the detection label conjugate.

[0350] This resulting complex of detection label: target analyte leaves the pad by capillary action and begins to migrate along the nitrocellulose membrane. When the complex reaches the analyte specific antibody, that is bound to the nitrocellulose membrane, a binding event occurs resulting in the immobilization of the complex and a visible test line result which is proportional to amount of analyte present.

[0351] If the analyte of interest is not present, then no complex will form and there will be no visible test line.

[0352] The test system incorporates a visual control line which is presented as a second visual line on the nitrocellulose membrane. The control line will be visible if sample has been added to the test, regardless of if target analyte is present in the sample. The control line, in this case a goat anti mouse antibody, is dried onto the nitrocellulose upstream of the analyte specific FITC antibody material. This control line will bind the detection label conjugate as it migrates though the test confirming that liquid has been added to the test and also that the detection label conjugate has migrated through the nitrocellulose strip. PV ref: 84489PC01

[0353] 38

[0354] A second pad is fixed to the opposite end of the nitrocellulose to the conjugate pad. This pad acts as a sink which draws the liquid through the test allowing the sample and detection label conjugate to clear through the nitrocellulose.

[0355] Example 13 - Half dipstick testing with striped membranes.

[0356] Aim of study

[0357] We aimed to determine pore sizes that would allow Rolling Circle Products (RCPs) to travel up a lateral flow strip and determine if either an Anti-Biotin or Anti-FITC test line would be suitable for signal detection. Half dipsticks were hand laminated using Grade 222 20 mm grade absorbance pad and three nitrocellulose (NC) membranes. Membranes CN95, CN140 and CN180 were selected for their different membrane flow rates (Table 2), and striped with 1 mg / mL Anti-biotin or 1 mg / mL anti-FITC; no control line was striped as this experiment was for signal detection only.

[0358] Methods

[0359] Samples supplied by VPCIR (Table 1) were diluted 1 : 10 and the line scores were evaluated using a test score card and photographed. A sequential method, as described in figure 12, was used to test the half dipsticks (Table 3).

[0360] Table 1: Summary and composition of sample supplied by VPCIR.

[0361] Table 2: Details of membranes selected for evaluation in VPCIR project. PV ref: 84489PC01

[0362] 39

[0363] Table 3: Summary of sequential method including contents required for each half dipstick and time within each well (mins).

[0364] Results

[0365] The experiment found some false positives present on the anti-biotin half dipsticks. False positives caused by non-specific binding (NSB) were observed with sample 1 on all conditions as evidenced by the test scores above 1 as seen on figure 13. CN140 produced the highest NSB and CN95 produced the lowest NSB. The greatest differential was observed with CN180, the negative control had an average test score of 3 whereas sample 2 and 4 scored 6. CN140 showed less differentiation between samples. In addition, there was inconsistencies with CN95. Sample 4 had the highest signal throughout all conditions.

[0366] Anti-FITC half dipsticks with CN95 membrane showed the least amount of NSB so was selected for further testing, as evidenced by the reduction in test score seen in figure 14. Initial testing with CN95 demonstrated differentiation between sample 1 (negative) and sample 2 (positive). Less differentiation was observed with sample 3 and 4, as expected in absence of FAM / FITC on the RCPs. These results suggest Anti-FITC test line assists orientation of RCPs for binding to Antibiotin conjugate.

[0367] The optical density (OD) of the gold conjugate was reduced to 0.5 to determine if the NSB could be reduced. Overall reduction of score across all conditions observed and NSB was still present on sample 1 as seen on figure 15. Condition with CN180 saw reduced differentiation between samples. CN95 showed the greatest differentiation between samples but the line quality was inferior.

[0368] Little differentiation between samples on condition with NC180 membrane, better differentiation on conditions with CN140 and CN95 membranes. Unexpected signal PV ref: 84489PC01

[0369] 40 was still observed with sample 3 and 4, these samples contain no FITC (figure 16).

[0370] Conclusion

[0371] Anti-biotin sandwich (Anti-biotin test line and anti-biotin conjugate gold) and anti- FITC: Anti-biotin format can detect RCPs present in the VPCIR samples. Overall, NSB was present but differentiation between samples was demonstrated. Anti- FITC line appeared to improve signal for sample 2.

[0372] Example 14 - Half Dipstick Testing with striped membranes, Combination. Aim of study

[0373] We aimed to continue investigation into most optimal membrane to allow RCPs to travel up a lateral flow strip and determine if either Anti-FITC or Anti-Biotin test line would provide better signal intensity. The same batch of half dipsticks from example 13 was used to test in example 14. Half dipsticks incorporating the three types of NC membranes (Table 2) were tested and compared with combination method (figure 12), an alternative to sequential method.

[0374] Methods

[0375] Samples 1-4 were tested with the anti-Biotin half dipsticks and samples 1-2 were tested with anti-FITC. Samples supplied by VPCIR (Table 1) were diluted 1: 10 and the line scores were evaluated using a test score card (ART114 VI.00) and photographed. Combination method was used to test the half dipsticks (Table 4).

[0376] Table 4: Summary of combination method including contents required for each half dipstick and time within each well

[0377] Results

[0378] Similar differentiation was observed between samples across all conditions, membrane speed did not affect the test score. There was a reduction in differentiation between sample 1 and 2, 3 and 4 compared to the sequential method results along with overall signal intensity. Test score of samples 2, 3 and 4 was lower across all conditions. In addition, false positives caused by NSB were still present (figure 17). PV ref: 84489PC01

[0379] 41

[0380] There was similar differentiation between samples 1 and 2 across all conditions, test score showed an average of 1 score difference. The NSB was still present across all conditions; sample 1 produced a test score of 1 or above for all conditions. Condition with CN95 membrane showed the lowest amount of NSB but greater signal intensity was observed with membranes CN180 and CN140, these membranes run slower and present increased binding time (figure 18).

[0381] Conclusion

[0382] Anti-biotin sandwich and anti-FITC: anti-biotin format can still detect signal using VPCIR samples in combination format, RCPs are successfully binding to the test line. However, combination testing has reduced differentiation between samples. Sequential is preferential for future testing.

[0383] Overall, initial stages of investigation established the anti-biotin gold conjugate could successfully bind to biotin and FAM labelled RCPs and signal can be detected using both formats. The RCPs could travel via capillary flow through the lateral flow test strip using CN180, 140 and 95 and be captured at the test line. Superior differentiation between samples was observed with CN95. There was some differentiation between biotin FAM incorporated samples and negative / RCP only in example 13 and 14. RCP was binding to conjugate and the conjugate: RCP complex was flowing in both test systems. No clear difference in test response was seen between CN95 and CN180. CN140 was ruled out in initial studies as it showed less differentiation between samples.

[0384] FAM annealing was successful as positive test line signal was observed with RCPs that was not present with probe alone. Anti-FITC sandwich system was successful with greater test line signals for elevated concentration of FAM probe. Anti-FITC sandwich showed greater test line signal than anti-biotin sandwich but poorer line quality. Dual annealing successful and the dual sandwich system was successful with both orientations of test line and conjugate evaluated. Sensitivity could be improved by increasing T-Line or conjugate concentration in the test system.

[0385] Example 15 - RCA products made from circles with different sequences can be detected in a lateral flow half-dipstick format.

[0386] Aim of study

[0387] We aimed to investigate if different DNA sequences would behave differently on a lateral flow half dipstick. We used a half dipstick with a test line functionalized PV ref: 84489PC01

[0388] 42 with anti-FITC antibodies and investigated the detection of RCA products generated from circles made from SEQ ID NO: 1 or SEQ ID NO: 7. The RCA products were labelled with a single FAM coming from a FAM end-labelled primer (SEQ ID NO: 15) and biotin that was incorporated by using biotin-conjugated dCTPs.

[0389] Using circles generated from the DNA substrates for mTOPl (SEQ ID NO: 1) and pTOPl (SEQ ID NO: 7), we showed that RCA products labelled with a 5'-FAM (by using a FAM functionalized primer. SEQ ID NO: 15) and with biotin (incorporated in the RCA products by using biotinylated dCTPs) could migrate in the membranes, bind to the test line and be labelled. This was not obvious as the RCA products generated from the two substrate sequences are bulky DNA structures. We demonstrate for the first time that the detection of enzyme biomarkers exemplified by but not limited to mTOPl and pTOPl can be read by using a lateral flow device combined with colorimetric readout.

[0390] Methods

[0391] The circles were prepared as described in example 3 for mTOPl or as in example

[0392] 9 for pTOPl and digested with exonucleases as described before.

[0393] The RCA was performed in solution in a buffer containing 50 mM Tris-HCI pH 7.5,

[0394] 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT supplemented with 0.2 pg BSA, 1 mM dNTP, 0.1 mM biotin dCTP, luM of FAM-anti ID33 primer (SEQ ID NO: 15) and 1 unit of Phi29 polymerase for 2 hours at 37°C.

[0395] The RCA products (RCPs) were filtered with microspin column (Cytiva#GE28- 9034-08) following manufacturer instruction.

[0396] For the half dipstick, 5 ul of RCPs were mixed with 15 ul of PBST and inserted in a well.

[0397] 20 ul of gold-nanoparticles conjugated with antibiotin antibody at OD 3 were applied to a second well and finally 20 ul of PBST were applied to a third well. Dipsticks containing anti-FITC antibody in the test line and anti-mouse antibody in the control line were immersed in the first well with RCPs for 5 minutes, followed by 5 minutes in the gold nanoparticles in the second well, followed by a wash in PBST in the third well for 5 minutes. The dipsticks were then airdried for 15 minutes and the lines scored by the use of a score card.

[0398] Results

[0399] RCA products generated from sequence 1 or 18 both resulted in a detection score of 6-7 as shown in figure 21. PV ref: 84489PC01

[0400] 43

[0401] Conclusion

[0402] The sequence differences between SEQ ID NO's: 1 and 7 do not affect the result of the lateral flow half dipstick readout format. This is not obvious as the sequence can affect the structure of RCA products.

[0403] Example 16 - Optimization of the lateral flow dipstick format Aim of study

[0404] We aimed to investigate how the lateral flow assay can be improved.

[0405] Methods

[0406] It is hypothesized that phi-29 primer elongation using unreacted substrate as a template creates double-stranded DNA products labelled with both FAM and biotin. They may travel faster in the membrane than the RCA products and compete with those for binding to test line. In top of that they become labelled with the antibiotin antibody gold-conjugated dye and result in background signal in both samples.

[0407] A solution is to make the following adaptions to the assay

[0408] 1) Add a capture moiety to the 5'-end or 3'-end of the oligonucleotide to be circularized, in such a way that when the enzyme in question cleaves the oligonucleotide in the process of generating a circle, the capture moiety is removed. The capture moiety should be at the 5'-end for mycobacteria substrates and at the 3'-end for the other substrates that all target type IB topoisomerases. The skilled person will know how to design such substrates (see e.g. WO2024 / 094858 Al).

[0409] 2) Insert a filter line containing an anti-capture moiety (such as a FICT antibody) that will trap unreacted substrate that acted as a template for primer elongation by phi29 before they reach the test line containing anti-DIG antibody or anti-biotin antibodies.

[0410] The capture moiety should be different from the label being incorporated by the RCA reaction (such as biotin).

[0411] Example 17 - Detection of mTOPl in clinical samples i.e. saliva from confirmed TB-positive individuals

[0412] Aim of study PV ref: 84489PC01

[0413] 44

[0414] Another solution to high background could be to improve the exonuclease digestion of the substrate mixture after circularization. To test this, circles were made using saliva from individuals with confirmed TB at lower NaCI concentrations as this allows for exonuclease digestion at lower NaCI concentrations (note, exonucleases are inhibited by NaCI concentrations >100 mM).

[0415] Methods

[0416] The circles were obtained by using saliva from one individual infected with TB (51, GenXpert confirmed) and one negative (6, GenXpert confirmed). The circularization reactions were performed by incubating the extracted saliva with 40 pmol of substrate SEQ ID NO: 1 in the presence of a buffer containing 10 mM Tris-HCI (pH 7.5), 30mM MgCh, ImM DTT and different concentrations of NaCI (50, 100, 200, 400 and 800 mM) for 3 hours at 37 °C. After exonuclease digestion, the RCA and dipsticks were performed as described in example 22.

[0417] Results

[0418] We observed more signal from TB-positive saliva with decreasing NaCI concentration while the background signal from TB-negative samples decreased with decreasing NaCI concentration probably because of more effective exonuclease digestion (figure 22).

[0419] Conclusion

[0420] By decreasing the NaCI in the circle reaction it is possible to distinguish between saliva from TB-positive and TB-negative individuals. The NaCI optimum for the lateral flow setup was 50 mM NaCI. This was surprising as it is far from the NaCI optimum observed the example 6.

[0421] Example 18 - Validation of the Pasodect technology combined with lateral flow for detection of tuberculosis in saliva, tongue swabs and sputum in Sub-Saharan Africa.

[0422] Aim of study

[0423] Matching sputum, saliva and tongue swabs from confirmed tuberculosis positive or negative individuals were analyzed by Pasodect in Bissau (Guinea Bissau) to validate the technology using clinical samples in an African context.

[0424] Method

[0425] 0.5 mL of Saliva or sputum was used directly to make extracts, the content from the tongue swab was dissolved in 0.2 mL of 10 mM Tris-HCI (pH 7.5) and 50 mM NaCI. Extracts from clinical samples were prepared as described in example 4 and PV ref: 84489PC01

[0426] 45 incubated with 40 pmol of oligonucleotide sequence 1 in a buffer containing 10 mM Tris-HCI (pH 7.5), 400 mM NaCI, 30mM MgCh, ImM DTT for 30 min. at 37 °C and analyzed as described in example 22 to generate circles and digested with exonucleases as described before.

[0427] The RCA was performed in solution in a buffer containing 50 mM Tris-HCI pH 7.5, 10 mM MgCh, 10 mM (NH4)2SC>4, 4 mM DTT supplemented with 0.2 pg BSA, 1 mM dNTP, 0.1 mM biotin dCTP, O.luM of FAM-anti ID33 primer and 1 unit of Phi29 polymerase for 1 hour at 37°C.

[0428] For the half dipstick, 5 ul of RCPs were mixed with 15 ul of PBST and inserted in a well. 20 ul of PBST was applied to a second well. 20 ul of gold-nanoparticles conjugated with antibiotin antibody at OD 3 were applied to a third well and finally 20 ul of PBST were applied to a fourth well.

[0429] Dipsticks containing anti-FITC antibody in the test line and anti-mouse antibody in the control line were immersed in the first well with RCPs for 5 minutes, followed by 5 minutes in the second well with PBST, 5 minutes in the third well with the gold nanoparticles, followed by a wash in PBST in the fourth well for 5 minutes. The dipsticks were then airdried for 15 minutes and the lines scored by the use of a score card.

[0430] Result

[0431] When analysing tongue swabs, we obtained a Sensitivity 25 / 27=92.6%, and specificity 13 / 13 = 100% (with two false negatives), while the sensitivity and specificity when saliva was less (Figure 23, bottom). Due to unavailability of sputum from negative individuals it was not possible to calculate sensitivity / specificity from this sample material. For comparison a picture of typical result on the LF sticks is also shown in Figure 23 (top).

[0432] Conclusion

[0433] The assay is working in all sample materials, but the signal-to-noise ratio (difference between TB positive and TB negative samples) and sensitivity / specificity are better when using swabs compared to saliva. The lack of sputum from TB-negative individuals makes it impossible to conclude much on this sample material.

[0434] Example 19 - Validation of the Pasodect technology combined with lateral flow for detection of malaria in saliva collected in Sub-Saharan Africa. Aim of study PV ref: 84489PC01

[0435] 46

[0436] Saliva from confirmed malaria positive or negative individuals were analyzed by Pasodect in to validate the technology.

[0437] Method

[0438] Active pTOPl was extracted from 0.2 mL of Saliva kept at 4°C by mixing with lxReporter lysis buffer (Promega, E3971) for 10 minutes on ice. Thereafter circles were prepared as described in example 9 using 4 ul of extract or 1 ng / pL pTOPl (as a control). Thereafter the RCA was performed in solution in a buffer containing 50 mM Tris-HCI pH 7.5, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT supplemented with 0.2 pg BSA, 1 mM dNTP, 0.1 mM biotin dCTP, 1 uM of anti ID33 primer (SEQ ID NO: 14) and 0.2 uM of FAM-TOPO primer (SEQ ID NO: 17) (branching oligo), and 1 unit of Phi29 polymerase for 1 hour at 37°C.

[0439] The readout using half-dipstick LFA was performed as described in example 18.

[0440] Results

[0441] 7 out of the 9 positive samples collected in Gabon and transported to Denmark gave a score higher than negative samples when using two primers for RCA as described above (Figure 24). The positive control gave a score higher than the negative control.

[0442] Conclusion

[0443] It is possible to detect malaria in saliva using the Pasodect method described above. However, transportation of samples may affect sensitivity.

[0444] Sequence listing PV ref: 84489PC01

[0445] 47 PV ref: 84489PC01

[0446] 48

[0447] Bold marks STS; Italic indicates primer-binding site.

[0448] Note to SEQ ID NO: 12: Reduced in G's to minimize background from uncircularized substrate. Items of the invention

[0449] Methods

[0450] 1. A method for determining whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases, the method comprising a) having a sample, such as a saliva sample or tongue swab, provided; b) providing a single stranded DNA oligonucleotide (1) comprising:

[0451] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl)

[0452] (2);

[0453] - a primer-annealing element, complementary to a DNA oligonucleotide (4); and PV ref: 84489PC01

[0454] 49 c) incubating in solution, preferably in the presence of Mg2+ions, the biological sample of step a) with the oligonucleotide (1) from step b), wherein if the DNA modifying enzyme, such as mTOPl (2) is present in the sample from step a), the single stranded DNA oligonucleotide of step b) is circularized (3); d) hybridizing the oligonucleotide (1) from step c) to the DNA oligonucleotide (4); e) determining the level of circularized oligonucleotides in the sample from step c) using Rolling Circle Amplification (RCA) with incorporation of labelled nucleotides, preferably the label is a first capture moiety, such a biotin moiety, such as biotin-dCTP; f) comparing said determined level to a reference level; and g) determining that if said level is above said reference level it is indicative of the sample comprising the pathogen; or if said reference level is equal to or below said reference, it is indicative of the sample not comprising the pathogen.

[0455] 2. The method according to item 1, wherein the pathogen is selected from the group consisting of Mycobacterium spp, plasmodium spp, and poxviruses, such as smallpox, cowpox, horsepox, camelpox, and mpox, Leishmania spp, and trypanosoma spp.

[0456] 3. The method according to item 1 or 2, wherein the Mycobacterium spp is the Mycobacterium tuberculosis complex, preferably the human Mycobacterium tuberculosis complex.

[0457] 4. The method according to item 1, wherein the pathogen is selected from the group consisting of Listeria spp., e. coll and salmonella spp.

[0458] 5. The method according to any of the preceding items, wherein the sample is a biological sample, such as selected from the group consisting of saliva, tongue swab, sputum, blood plasma, blood serum, urine, cell smear, faeces, cerebrospinal fluid, and a biopsy, preferably being a saliva sample or tongue swab, more preferably a lysed saliva sample or tongue swab.

[0459] 6. The method according to any of the preceding items, wherein the sample is a saliva sample or tongue swab. PV ref: 84489PC01

[0460] 50

[0461] 7. The method according to any of items 1-4, wherein the sample is selected from the group consisting of a food sample, a water sample, an environmental sample, such as liquid, water, soil, air, and plant samples, such as seeds or said sample is selected from the group consisting of animal feed or food for human consumption, such as meat and meat products, dairy products, such as milk, cream, fermented milk products, cheeses and butter, plants, such as vegetables and fruit, fish and fish products, egg and egg products, condiments and spices.

[0462] 8. The method according to any of the preceding items, wherein the sample is lysed, preferably lysis is performed by i) freeze and thawing the sample, preferably followed by vortexing with a solid material, such as glass beads; ii) bead beating; and / or iii) using lytic mycobacteriophages.

[0463] 9. The method according to any of the preceding items, wherein the subject is a mammal preferably a human. >

[0464] 10. The method according to any of the preceding items, wherein the binding element of the single stranded oligonucleotides (1) of step b) is selected from the groups consisting of:

[0465] • 5'-CGCTTG-3', 5'-CGCTTC-3', 5'-CTCTTG-3', and 5'-CTCTTC-3' (mTOPl); or

[0466] • CCCTT (mPoxTOPl)

[0467] 11. The method according to any of the preceding items, wherein the binding element of the single stranded oligonucleotides (1) of step b) is located within the first 50 nucleotides, preferably within the first 40 nucleotides, more preferably within the first 30 nucleotides, most preferably within the first 20 nucleotides of the 5'-end.

[0468] 12. The method according to any of the preceding items, wherein the sequence element of the oligonucleotide (1) 5' to primer-annealing element, has

[0469] • a percentage of nucleotides complementary to the labelled nucleotides to be incorporated in step e) in the range 0-10%, such as 0-5%, such as 0- 2% preferably 0%; and / or

[0470] • a number of nucleotides complementary to the labelled nucleotides to be incorporated in step e) in the range 0-5, such as 0-3, such as 0-2, preferably 0-1 and or more preferably 0. PV ref: 84489PC01

[0471] 51

[0472] 13. The method according to any of the preceding items, wherein the sequence element of oligonucleotide (1) 5' to the primer-annealing element, has a length in the range 5-30 nucleotides, such as 10-20 nucleotides.

[0473] 14. The method according to any of the preceding items, wherein the primerannealing element of the single stranded oligonucleotides (1) of step b) is complementary to a DNA oligonucleotide (4) attached to a solid support (5), such as a glass slide.

[0474] 15. The method according to item 14, wherein the oligonucleotide (4) attached to a solid support (5) is modified with an amine, preferably at the 5'-end.

[0475] 16. The method according to any of the preceding items 1-13, wherein the oligonucleotide (4) comprises a third capture moiety, such as a FAM moiety or FITC moiety, preferably the label is at the 5'-end, preferably said third capture moiety being different from the first capture moiety and second capture moiety.

[0476] 17. The method according to item 16, wherein the DNA oligonucleotide (4) is in solution and / or not being bound to a solid support.

[0477] 18. The method according to any one of the preceding items, wherein the primer annealing element of the single stranded oligonucleotide (1) of step b) is at least 25 nucleotides long, preferable at least 20 nucleotides long, more preferably at least 18 nucleotides long, such as having a length in the range 18-25 nucleotides.

[0478] 19. The method according to any of the preceding items, wherein the single stranded DNA oligonucleotide (1) of step b) has a length in the range 80-120 nucleotides, such as 90-110, preferably in the range 95-105 nucleotides, such as 98-102.

[0479] 20. The method according to any of the preceding items, wherein the single stranded oligonucleotide (1) of step b) is selected from the group consisting of SEQ ID NO': 1-12.

[0480] 21. The method according to any of the preceding items, wherein the oligonucleotide (1) comprises a second capture moiety at the 5'-end or the 3'-end, said second capture moiety being different from the first capture moiety and third capture moiety, such as the second capture moiety being FAM or FITC.

[0481] 22. The method according to item 21, wherein the second capture moiety is positioned in the first oligonucleotide allowing it to be cleaved of after circularization of the oligonucleotide (1) by enzymatic action of the DNA modifying enzyme. > PV ref: 84489PC01

[0482] 52

[0483] 23. The method according to any of the preceding items, wherein an exonuclease treatment step is included after step c) and preferably before step d), preferably the exonucleases are inactivated, preferably by heat-inactivation, before hybridization of the DNA oligonucleotide (1) to the DNA oligonucleotide (4).

[0484] 18. The method according to any of the preceding items, wherein step c) is performed with a concentration of MnCh below 1 mM, such as below 0.1 mM MnCh, preferably below 0.01 mM MnCh, and more preferably substantially in the absence of MnCh. or in the absence of MnCh.

[0485] 24. The method according to any of the preceding items, wherein in step c) Mg2+ions are present in the range 2-50 mM, preferably in the range 10-40 mM, more preferably in the range 20-40 mM, or in the range 25-35 mM.

[0486] 25. The method according to any of the preceding items, wherein in step c) Na+ions are present in an amount of 50-3000 mM, such as at a concentration in the range 100-2000 mM, preferably 200-1000 mM, more preferably 300-500 mM or wherein in step c) Na+ions are present in an amount of 0-3000 mM, such as at a concentration in the range 0-2000 mM, preferably 0-1000 mM, more preferably 0- 100 mM or 10-100 mM.

[0487] 26. The method according to any of the preceding items, wherein in step c) the solution contains

[0488] • below 0.1 mM Mn2+, preferably below 0.01 mM Mn2+;

[0489] • in the range 10-40 mM Mg2+, preferably in the range 20-40 mM Mg2+; and

[0490] • in the range 200-1000 mM Na+; or contains

[0491] • below 0.1 mM Mn2+, preferably below 0.01 mM Mn2+;

[0492] • in the range 10-40 mM Mg2+, preferably in the range 20-40 mM Mg2+; and

[0493] • in the range 0-100 mM Na+or 10-100 mM Na+.

[0494] 27. The method according to any of the preceding items, wherein in step e) biotin-labelled nucleotides are used, such as biotin-dCTP or fluorescently labelled nucleotides, or labelled nucleotides recognizable by an antibody.

[0495] 28. The method according to any of the preceding items, wherein in step e) the polymerase phi29 is used for RCA. PV ref: 84489PC01

[0496] 53

[0497] 29. The method according to any of the preceding items, wherein the level of circularized oligonucleotides in the sample in step e), is determined by an assay selected from a chemiluminescence assay and a colorimetric assay.

[0498] 30. The method according to any of the preceding items, wherein the level of circularized oligonucleotides in the sample in step e), is determined by chemiluminescence assay, wherein HRP conjugated with an anti-biotin antibody and / or HRP conjugated with a streptavidin is bound to the RCA product, containing biotin-labelled nucleotides, and signals are generated by adding ECL, followed by detection.

[0499] 31. The method according to item any of the preceding items 1-29, wherein the level of circularized oligonucleotides in the sample in step e), is determined by a colorimetric assay, wherein HRP conjugated with an anti-biotin antibody or HRP conjugated with a streptavidin is bound to the RCA product, containing biotin- labelled nucleotides, and signals are generated by adding a chromogenic substrate, such as TMB (3,3',5,5'-tetramethylbenzidine), and an oxidizing agent, such as hydrogen peroxide (H2O2), followed by detection.

[0500] 32. The method according to item 31, wherein the chromogenic substrate is selected from the group consisting of TMB, ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine dihydrochloride), DAB (3,3'-diaminobenzidine), and BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium), preferably being TMB.

[0501] 33. The method according to any of the preceding items, wherein the level of RCA product is determined in a lateral flow assay.

[0502] 34. The method according to item 33, wherein the lateral flow device comprises a membrane material comprising nitrocellulose, such as CN95, CN140 or CN180DX.

[0503] 35. The method according to item 33, wherein the lateral flow assay comprises a capture element adapted for capturing uncircularized oligonucleotides (1) elongated by the polymerase, such as through a capture moiety present on the 5'- end or the 3'end of the oligonucleotides (1), preferably being adapted to capture the uncircularized oligonucleotides (1) elongated by the polymerase before they reach the test line in the lateral flow assay, preferably the test line contains anti- DIG antibodies or anti-biotin antibodies.

[0504] 36. The method according to any of the preceding items, wherein in step d) also includes the addition of a branching oligonucleotide, such as SEQ ID NO: 17. PV ref: 84489PC01

[0505] 54

[0506] Step f

[0507] 37. The method according to any of the preceding items, wherein the reference level of step f) is determined in a subject or a group of subjects without a corresponding pathogenic infection, such as a Mycobacterium tuberculosis infection.

[0508] 38. The method according to any of the preceding items, wherein the reference level of step f) is determined using the same method as used to determine the level of circularized DNA in step e).

[0509] 39. The method according to item 1, wherein if said level is above said reference level it is indicative of said subject being infected with the pathogen, such as with Mycobacterium spp; or if said reference level is equal to or below said reference, it is indicative of said subject not being infected with the pathogen, such as with Mycobacterium spp.

[0510] Kit

[0511] 40. A kit of parts comprising a) a single stranded DNA oligonucleotide (1) comprising:

[0512] - a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase I enzyme (mTOPI) (2);

[0513] - a primer-annealing element, complementary to a DNA oligonucleotide (4); b) a labelled DNA oligonucleotide (4), suitable for acting as a primer for an RCA reaction of a circularized version of the single stranded DNA oligonucleotide (1); and c) a lateral flow device adapted to detect an RCA product comprising labelled nucleotides, such as biotins; d) optionally reaction components, such as a lysis buffer, exonuclease solution, RCA solutions and / or detection solutions; and e) optionally, a gel filtration column, adapted for removing excess reagents; and f) optionally a branching oligo, such as SEQ ID NO: 17. PV ref: 84489PC01

[0514] 55

[0515] 41. The kit of parts according to item 40, wherein the lateral flow device adapted to detect an RCA product comprises a capture element adapted for capturing uncircularized oligonucleotides (1) that act as template for primer elongation by the polymerase, such as through a capture moiety present on the 5'-end or the 3'-end of the oligonucleotides (1), preferably being adapted to capture the uncircularized oligonucleotides (1) that template primer elongation by the polymerase before they reach the test line in the lateral flow assay, preferably the test line contains anti-DIG antibodies or anti-biotin antibodies.

[0516] Uses

[0517] 42. use of the kit of parts according to item 40 or 41 for determining, whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases.

[0518] 43. The use according to item 42, for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with Mycobacterium spp, preferably the Mycobacterium tuberculosis complex.

Claims

PV ref: 84489PC0156Claims1. A method for determining whether a sample comprises a pathogen or not, such as for determining whether a subject is likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases, the method comprising a) having a sample, such as a saliva sample or tongue swab, provided; b) providing a single stranded DNA oligonucleotide (1) comprising:- a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase 1 enzyme (mTOPl) (2);- a primer-annealing element, complementary to a DNA oligonucleotide (4); and c) incubating in solution, preferably in the presence of Mg2+ions, the biological sample of step a) with the oligonucleotide (1) from step b), wherein if the DNA modifying enzyme, such as mTOPl (2) is present in the sample from step a), the single stranded DNA oligonucleotide of step b) is circularized (3); d) hybridizing the oligonucleotide (1) from step c) to the DNA oligonucleotide (4); e) determining the level of circularized oligonucleotides in the sample from step c) using Rolling Circle Amplification (RCA) with incorporation of labelled nucleotides, preferably the label is a first capture moiety, such a biotin moiety, such as biotin-dCTP; f) comparing said determined level to a reference level; and g) determining that if said level is above said reference level it is indicative of the sample comprising the pathogen; or if said reference level is equal to or below said reference, it is indicative of the sample not comprising the pathogen.

2. The method according to claim 1, wherein the pathogen is selected from the group consisting of Mycobacterium spp, plasmodium spp, and poxviruses, such as smallpox, cowpox, horsepox, camelpox, and mpox, Leishmania spp, andPV ref: 84489PC0157 trypanosoma spp, preferably from the human Mycobacterium tuberculosis complex.

3. The method according to any of the preceding claims, wherein the sample is a saliva sample or tongue swab.

4. The method according to any of the preceding claims, wherein the oligonucleotide (1) comprises a second capture moiety at the 5'-end or the 3'-end, said capture moiety preferably being different from the first capture moiety and the third capture moiety, such as the second capture moiety being FAM or FITC.

5. The method according to claim 4, wherein the second capture moiety is positioned in the first oligonucleotide allowing it to be cleaved of after circularization of the oligonucleotide (1) by enzymatic action of the DNA modifying enzyme.

6. The method according to any of the preceding claims, wherein the oligonucleotide (4) comprises a third capture moiety, such as a FAM label or FITC label, preferably the label is at the 5'-end.

7. The method according to any of the preceding claims, wherein the DNA oligonucleotide (4) is in solution and / or not being bound to a solid support.

8. The method according to any of the preceding claims, wherein the single stranded oligonucleotide (1) of step b) is selected from the group consisting of SEQ ID NO': 1-12.

9. The method according to any of the preceding claims, wherein an exonuclease treatment step is included after step c) and preferably before step d), preferably the exonucleases are inactivated, preferably by heat-inactivation, before hybridization of the DNA oligonucleotide (1) to the DNA oligonucleotide (4).PV ref: 84489PC015810. The method according to any of the preceding claims, wherein in step e) biotin-labelled nucleotides are used, such as biotin-dCTP or fluorescently labelled nucleotides, or labelled nucleotides recognizable by an antibody.

11. The method according to any of the preceding claims, wherein the level of circularized oligonucleotides in the sample in step e), is determined by an assay selected from a chemiluminescence assay and a colorimetric assay.

12. The method according to any of the preceding claims, wherein the level of RCA product is determined in a lateral flow assay.

13. The method according to any of the preceding claims, wherein in step d) also includes the addition of a branching oligonucleotide, such as SEQ ID NO: 17.

14. A kit of parts comprising a) a single stranded DNA oligonucleotide (1) comprising:- a binding element for the DNA modifying enzyme from the pathogen, such as a Strong Topoisomerase Site (STS) for Mycobacterium spp. DNA Topoisomerase I enzyme (mTOPI) (2);- a primer-annealing element, complementary to a DNA oligonucleotide (4); b) a labelled DNA oligonucleotide (4), suitable for acting as a primer for an RCA reaction of a circularized version of the single stranded DNA oligonucleotide (1); and c) a lateral flow device adapted to detect an RCA product comprising labelled nucleotides, such as biotins; d) optionally reaction components, such as a lysis buffer, exonuclease solution, RCA solutions and / or detection solutions; e) optionally, a gel filtration column, adapted for removing excess reagents; and f) optionally a branching oligo, such as SEQ ID NO: 17.

15. Use of the kit of parts according to claim 14 for determining, whether a sample comprises a pathogen or not, such as for determining whether a subject isPV ref: 84489PC0159 likely to be infected with a pathogen, said pathogen expressing DNA modifying enzymes capable of cleaving and ligating DNA, such as topoisomerases and integrases.

16. The use according to claim 15, for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with the pathogen, wherein the pathogen is selected from the group consisting of Mycobacterium spp, plasmodium spp, and poxviruses, such as smallpox, cowpox, horsepox, camelpox, and mpox, Leishmania spp, and trypanosoma spp, preferably from the human Mycobacterium tuberculosis complex.

17. The use according to claim any of claims 15-16, for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with Mycobacterium spp, preferably from the Mycobacterium tuberculosis complex.

18. The use according to claim 15 or 16, for determining, in a saliva sample or tongue swab, whether a subject is likely to be infected with plasmodium spp.

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