Methods and devices for multi-tagged nucleic acid amplification and detection

Abstract

The present disclosure describes methods, compositions, kits and uses for nucleic acid amplification and subsequent detection of nucleic acid amplification products using labeled nucleotides and differentially labeled probes or primers. In the present disclosure, the properties of recombinase and individually labeled probes are used. The disclosed methods, compositions and kits enable more sensitive and more specific detection of nucleic acid amplification products at low temperatures, thereby providing improved means for detecting nucleic acids in a low resource environment.

Description

Field of the Invention

[0001] The present invention relates to methods and systems for nucleic acid amplification, preferably isothermal nucleic acid amplification, such as recombinase polymerase amplification, using labeled nucleotides and differentially labeled probes or primers. In addition, the present invention relates to methods and systems for detecting amplified nucleic acids using recombinases, labeled probes, and optionally components (means) for regulating the binding of the recombinase or labeled probe to the amplified nucleic acids. The present invention also relates to the use of the methods and systems in healthcare, industry, and research. Background of the Invention

[0003] The development of isothermal nucleic acid amplification methods as an alternative to polymerase chain reaction (PCR) is progressing rapidly. Unlike PCR, isothermal nucleic acid amplification has the advantage that it can be performed at a constant and optionally ambient temperature and does not require cyclic temperature changes. In addition, isothermal nucleic acid amplification and subsequent detection methods require fewer resources and are easier to perform, in many cases without the use of laboratories or professionals.

[0004] One such isothermal amplification method is recombinase polymerase amplification (RPA) (WO9117267A1 and Piepenburg et al., PLoS Biol. 2006 Jul; 4 (7): e204). The RPA method uses a recombinase and a DNA polymerase, as well as one or more oligonucleotide primers and optional DNA binding-promoting proteins to achieve DNA amplification. The recombinase-primer complex is formed in cooperation with a cofactor, which unwinds the two strands of the DNA duplex at a DNA sequence complementary to the primer. The strand displacement polymerase then extends the primer, thereby amplifying the target sequence. One of the advantages of RPA over PCR is that the efficacy of RPA is less affected by compounds that can be found in biological samples and that typically inhibit PCR.

[0005] Like PCR and isothermal amplification methods, RPA has been used for the amplification and detection of DNA and RNA of various organisms. The detection of nucleic acids is important in many industries such as the healthcare, food and pharmaceutical industries because determining the absence or presence of these nucleic acids provides valuable information. For example, the absence of specific nucleic acids may be necessary for the production of certain drugs or foods, while the presence of pathogen nucleic acids helps in the diagnosis of diseases or determines the spread of these pathogens. However, a disadvantage of RPA is the generation of non-specific amplification products, which depends on the primers used for amplification. General guidelines for primer design are known. However, they are inadequate, and no primer design software can accurately predict the primer pairs that will perform best. Therefore, improved methods are needed to detect nucleic acids.

[0006] Amplification by PCR and isothermal amplification methods (e.g., RPA) can be monitored by various detection methods, either via endpoint detection after amplification or via real-time detection during amplification, and can be achieved by using probes (e.g., oligonucleotides and / or fluorescent probes) (Lobato and O'Sullivan, Trends Analyt Chem. 2018 Jan; 98: p19-35). Oligonucleotide probes contain reference sequences, and thus these probes can be used to verify the exact specificity of the amplification product to reduce the chance of nonspecific detection.

[0007] End-point detection is usually performed via lateral flow devices or strips, while real-time detection is usually performed via fluorescent probes and fluorometers. Popular detection methods for RPA are TwistDx’s Kits are provided which involve the use of labeled oligonucleotide probes and nucleases targeting the probes (WO2010141940A1 and Piepenburg et al.). The technology uses probe-based DNA detection during recombinase polymerase amplification. The technology uses highly modified single-stranded DNA probes that are cleaved by restriction enzymes and emit fluorescence (Jia et al., Archives of Virology, 2020). Such probes can also be extended, essentially becoming a second primer that produces an amplified fragment of dsDNA visualized on a lateral flow strip (Zou et al., Journal of Virological Methods, 2022).

[0008] However, these methods have several disadvantages. For example, lateral flow strips typically use gold nanoparticles and antibodies, which increases the cost of the assay. The kit introduces a specific probe design, including multiple non-natural modifications and the use of specific nucleases. Due to the complexity of designing and screening the best probes, These aspects of the technology hinder the optimization of diagnostic assays. Real-time detection requires advanced readout equipment. These disadvantages are exacerbated in low-resource environments. Therefore, there is still a need for simple, cost-effective and / or improved detection methods for nucleic acid amplification reactions, especially isothermal amplification reactions such as RPA, especially for low-resource environments. Due to the complexity of RPA, it has been difficult to improve. However, since RPA is performed at low temperatures, there is still a need to improve the specificity of the method. SUMMARY OF THE INVENTION

[0010] The inventors were surprised to find that, compared with the prior art, an amplified DNA product (amplicon) having a target DNA sequence can be specifically detected via a simpler method. The present invention includes the use of sequence-specific labeled probes without the need for exogenous enzymes and expensive probe modifications. The labeled probe is incubated with the recombinase and the nucleic acid amplification product, and the resulting three-stranded DNA / recombinase complex is then detected via, for example, an antibody that is targeted and attached to a solid support (such as a lateral flow strip). The surprising discovery comes from the prior art, which indicates that it is impossible to detect the complex itself. EP0612352A1 discloses that a stable probe-dsDNA complex can only be formed when a double D loop is formed (containing a double-stranded probe, thereby forming a four-stranded DNA) and the recombinase is subsequently removed from the complex. US6132972A and US2003 / 044819A1 disclose that the probe-dsDNA complex should be cleaved with a nuclease (similar to Probe design) and deproteinized (e.g., by using a protease) for detection. Therefore, the present invention does not require enzymatic post-treatment for detecting triple-stranded DNA complexes, but only uses single-stranded labeled probes and recombinases. Screening for optimal probes can preferably be performed without chemical modification of the probe backbone, and can be performed, for example, on a simple agarose gel, where the intensity of the tail will be related to the strength of the probe binding to the target dsDNA. This significantly reduces the time and cost associated with probe screening.

[0011] In addition, the inventors have surprisingly found that compared with current methods in the art, amplified DNA products (amplicons) having a target DNA sequence can be detected with higher specificity and sensitivity. This is achieved by preferably incorporating into the amplicon more than one deoxynucleoside triphosphate (dNTP) monomer containing a first label (e.g., biotin) that binds to an amplicon of an oligonucleotide probe containing a nucleic acid sequence complementary to the target DNA sequence, and incorporating a second label (e.g., another hapten) that is different from the first label. The probe binds to the target DNA sequence with the help of a recombinase. The first label or the second label is located on the probe. If the first label (preferably multiple) is present on the probe, the second label is present on one of the primers used for amplification. The probe can then also contain a sequence (part) of one of the other primers used for amplification. When the second label is present on the probe, the first label is incorporated into the (preferably multiple) amplicon. The probe can then also contain a sequence (part) of one of the primers used for amplification. The probe and / or the recombinase can be added after the nucleic acid amplification reaction, or they can be added before or during the nucleic acid amplification reaction, thereby achieving a so-called one-pot reaction. See Figure 1 and Figure 2 To obtain a schematic diagram of the concept as described above. The oligonucleotide probe according to the present invention need not be complementary to the entire target DNA sequence, but may be complementary to a portion of the target DNA sequence, for example.

[0012] More than one first label enables easier detection of amplicons by various means (e.g., via lateral flow strips), thereby increasing the sensitivity of detecting the target DNA sequence, thereby reducing the components required for detection. The use of two different labels and probes increases the specificity of target DNA sequence detection because the probes containing the different labels target the target DNA sequence in the amplified nucleic acid product independently of the primers used to amplify the target DNA sequence. The recombinase enables the binding of the probe to be performed at low temperatures (e.g., 20 to 50° C.) without the use of advanced equipment.

[0013] Thus, the disclosed methods, compositions, and kits enable more sensitive and specific detection of nucleic acid amplification products at low temperatures, thereby providing improved means for detecting nucleic acids in low-resource settings.

[0014] Therefore, the object of the present invention is to provide a method for determining the presence or absence of a target deoxyribonucleic acid (DNA) sequence in a sample, the method comprising:

[0015] a. Contact the sample with:

[0016] a first primer and a second primer for amplifying a target DNA sequence;

[0017] Recombinase;

[0018] DNA-dependent DNA polymerase;

[0019] A deoxynucleoside triphosphate (dNTP) monomer containing a first label; and

[0020] an oligonucleotide probe comprising a nucleic acid sequence complementary to a target DNA sequence and a second label;

[0021] b. amplifying the target DNA sequence to produce a nucleic acid amplification product; and

[0022] c. Detect nucleic acid amplification products.

[0023] Another object is to provide a method for determining the presence or absence of a target DNA sequence in a sample, the method comprising:

[0024] a. Contact the sample with:

[0025] a first primer and a second primer for amplifying the target DNA sequence, wherein the second primer contains a second label and hybridizes to the first strand of DNA of the target DNA sequence;

[0026] Recombinase;

[0027] DNA-dependent DNA polymerase; and

[0028] an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label;

[0029] b. amplifying the target DNA sequence to produce a nucleic acid amplification product; and

[0030] c. Detect nucleic acid amplification products.

[0031] Another object of the present invention is to provide an ex vivo method for diagnosing a disease or condition, preferably an infectious disease, in a subject, comprising determining the presence or absence of a target DNA sequence associated with the disease or condition according to the present invention in a sample obtained from the subject, wherein the presence indicates a diagnosis of the disease or condition in the subject.

[0032] Another object is to provide a method for detecting a nucleic acid amplification product comprising a target DNA sequence, the method comprising:

[0033] a. contacting the nucleic acid amplification product with a recombinase and an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a label, thereby forming a D-loop comprising triple-stranded DNA, the probe and the recombinase in the nucleic acid amplification product; and

[0034] b. Detecting nucleic acid amplification products, including detection probes.

[0035] Another object is to provide a composition comprising:

[0036] A dNTP monomer containing a first label and an oligonucleotide probe containing a second label; or

[0037] A first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and is capable of hybridizing with a first strand of DNA of the target DNA sequence; and an oligonucleotide probe, the oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first label, preferably, wherein the probe is capable of binding or hybridizing with a first strand or a second strand of DNA of the target DNA sequence, the second strand being complementary to the first strand.

[0038] Yet another object is to provide a lateral flow device comprising a lateral flow strip, the lateral flow strip comprising:

[0039] - sample application zone;

[0040] - a reagent zone downstream of and in fluid communication with the sample application zone, wherein the reagent zone comprises a non-immobilized first binding agent bound to a first label;

[0041] - a test zone downstream of and in fluid communication with the reagent zone, wherein the test zone comprises an immobilized second binding agent bound to a second label; and

[0042] - a control zone downstream of the test zone and in fluid communication with the test zone.

[0043] Another object is to provide a kit of parts comprising:

[0044] a. Optionally, a first primer and a second primer for amplifying a target DNA sequence;

[0045] b. Recombinase;

[0046] c.DNA-dependent DNA polymerase;

[0047] d. a dNTP monomer containing a first label; and

[0048] e. an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a second label; and optionally

[0049] f. Lateral flow device or device according to the invention.

[0050] Another object is to provide a kit of parts comprising:

[0051] a. a first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and hybridizes to a first strand of DNA of the target DNA sequence;

[0052] b. Recombinase;

[0053] c. DNA-dependent DNA polymerase; and

[0054] d. an oligonucleotide probe comprising a nucleic acid sequence complementary to a target DNA sequence and one or more nucleotides containing a first label, preferably wherein the probe hybridizes to a first strand or a second strand of DNA of a target DNA sequence, the second strand being complementary to the first strand; and optionally

[0055] e. Lateral flow device or device according to the invention.

[0056] Yet another object is to provide a device or a kit of components according to the present invention for determining the presence or absence of a pathogen, preferably a human, animal or plant pathogen, or a target DNA sequence associated with a disease or disorder or the onset thereof, or for determining the presence or absence of a target DNA sequence in a diagnostic sample, a veterinary sample, a pharmaceutical sample, a cosmetic sample, a food sample, a beverage sample, a feed sample, an agricultural sample, a soil sample, an air sample, a water sample, a waste sample, an effluent sample or a sewage sample.

[0057] A brief description of the attached figure

[0058] Figure 1A schematic depiction of a first method of the present invention is shown.

[0059] Figure 2 A schematic depiction of a second method of the present invention is shown.

[0060] Figure 3 The effect of biotin-dCTP concentration on RPA yield is shown.

[0061] Figure 4 The effect of biotin-dUTP concentration on RPA yield is shown.

[0062] Figure 5 Shown is the effect of both biotin-dCTP and biotin-dUTP concentrations on RPA yield.

[0063] Figure 6 Evidence for D-loop formation on an agarose gel is shown.

[0064] Figure 7 Evidence for D-loop formation in ELISA is shown.

[0065] Figure 8 Shown is evidence for D-loop formation in a lateral flow assay.

[0066] Fig. 9 The effect of probe design on lateral flow assays is shown.

[0067] Fig.10 The specificity of the primer-probe and the probe with and without ATP-γ-S compared to the hapten-based strategy is shown.

[0068] Fig.11 It was shown that RPA amplicons can be directly added to an immobilized D-loop detection coupled to a RecA probe with high reproducibility and specificity.

[0069] Fig.12 Shown that DrRecA does not require ATP or ATPγS for strand exchange.

[0070] Fig.13 It was shown that bridge flocculation allowed visual detection of biotinylated DNA.

[0071] Fig.14 Stable D-loop formation of EcRecA and DrRecA is shown. DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention relates to two methods with a similar concept, but wherein the first and second labels are present on different components. The second label is different from the first label. Figure 1 An overview of the first method is shown, and Figure 2 An overview of the second method is shown.

[0074] The present invention relates to a first method for determining the presence or absence of a target deoxyribonucleic acid (DNA) sequence in a sample, the method comprising:

[0075] a. Contact the sample with:

[0076] a first primer and a second primer for amplifying a target DNA sequence;

[0077] Recombinase;

[0078] DNA-dependent DNA polymerase;

[0079] A deoxynucleoside triphosphate (dNTP) monomer containing a first label; and

[0080] an oligonucleotide probe comprising a nucleic acid sequence complementary to a target DNA sequence and a second label;

[0081] b. amplifying the target DNA sequence to produce a nucleic acid amplification product; and

[0082] c. Detect nucleic acid amplification products.

[0083] The present invention also relates to a second method for determining the presence or absence of a target deoxyribonucleic acid (DNA) sequence in a sample, the method comprising:

[0084] a. Contact the sample with:

[0085] a first primer and a second primer for amplifying the target DNA sequence, wherein the second primer contains a second label and hybridizes to the first strand of DNA of the target DNA sequence;

[0086] Recombinase;

[0087] DNA-dependent DNA polymerase; and

[0088] an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label;

[0089] b. amplifying the target DNA sequence to produce a nucleic acid amplification product; and

[0090] c. Detect nucleic acid amplification products.

[0091] Without being bound by theory, it is proposed that the recombinase according to the present invention and the probe together enable the formation of a D-loop in the nucleic acid amplification product at a sequence complementary to the probe, thereby forming a triple-stranded DNA. "D-loop" herein is understood to mean a DNA structure in which the two chains of a double-stranded DNA molecule are separated by a certain length and kept separated by the third chain of the DNA optionally containing the recombinase. This is also referred to as a single D-loop, which is different from the two chains of the double-stranded DNA molecule in which the two chains are separated by a certain length and kept separated by another two DNA chains, thereby forming a double D-loop of a four-stranded DNA. Therefore, the D-loop according to the present invention includes triple-stranded DNA. The recombinase can still be combined in the D-loop, preferably by a component for regulating the binding of the recombinase to DNA, or it can be dissociated. If the latter occurs, the probe should still be combined in the D-loop, preferably by a component for regulating the binding of the labeled probe to DNA. It is believed that the D-loop complex comprising the target DNA sequence of the probe and the amplification and the optional recombinase formed by the method according to the present invention has not been shown to be possible for detecting nucleic acid amplification products so far. Therefore, the inventors have surprisingly shown that it can be used in this way and applied to the reliable detection of nucleic acid amplification products. According to the present invention, the nucleic acid amplification product preferably comprises triple-stranded DNA, preferably DNA comprising the D-loop.

[0092] The inventors have shown that a nucleic acid amplification product comprising a D-loop comprising triple-stranded DNA, a probe and a recombinase is sufficiently stable to be detected by a detection probe. Therefore, step c of detecting the nucleic acid amplification product according to the first or second method of the present invention preferably comprises a nucleic acid amplification product comprising a D-loop comprising triple-stranded DNA, a probe and a recombinase, wherein detecting the nucleic acid amplification product comprises detecting, preferably in combination with, the probe.

[0093] Preferably, contacting the sample with the recombinase and / or the probe may be performed before, during or after step b of amplifying the target DNA sequence.

[0094] Herein, the terms "three-stranded DNA" and "DNA triplex" are used interchangeably. It should be understood that triple-stranded DNA herein refers to intermolecular DNA triplexes rather than intramolecular DNA triplexes. Intermolecular triplexes are formed between (triplex-forming) oligonucleotides (such as oligonucleotide probes according to the present invention) and target DNA sequences on double-stranded DNA (dsDNA). Intramolecular triplexes are formed when, for example, the third strand is part of a single strand that also contains dsDNA.

[0095] As used herein, the term "nuclease-free" should be understood to mean that there is no nuclease for forming a signal (or its precursor) for detection, for example, the formation of the signal is promoted in the absence of a nuclease. Similarly, the term "protease-free" as used herein should be understood to mean that there is no protease for forming a signal (or its precursor) for detection, for example, the formation of the signal is promoted in the absence of a protease. For example, when carrying out the method according to the present invention, nucleases or proteases are not used during any step of the method. Therefore, it is preferred that nucleases and / or proteases are not present in the kit and composition according to the present invention. As disclosed above and herein, the present invention does not require enzymatic post-treatment of nucleic acid amplification products (e.g., via nucleases or proteases) to detect triple-stranded DNA complexes, and only single-stranded labeled probes and recombinases are used.

[0096] The oligonucleotide probe according to the present invention is preferably single-stranded, preferably comprising single-stranded DNA. The oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a second marker according to the present invention is preferably single-stranded, preferably comprising single-stranded DNA. The oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first marker is preferably single-stranded, preferably comprising single-stranded DNA. The single-stranded nature of the oligonucleotide probe enables the formation of a D-loop comprising triple-stranded DNA according to the present invention together with a recombinase and a target DNA sequence.

[0097] The oligonucleotide probe comprising a nucleic acid sequence complementary to a target DNA sequence and a second label according to the present invention may comprise a partial nucleic acid sequence or the entire nucleic acid sequence of one of the primers used for amplification.

[0098] According to the present invention, the oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first label can include a partial nucleic acid sequence or a complete nucleic acid sequence of the first primer for amplification. The probe comprising a partial nucleic acid sequence or a complete nucleic acid sequence of one of the primers can promote the formation of the D ring in the nucleic acid amplification product. Preferably, the partial nucleic acid sequence includes at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the complete nucleic acid sequence of one of the primers for amplification or the first primer for amplification. Preferably, the partial nucleic acid sequence includes at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% of the complete nucleic acid sequence of one of the primers for amplification or the first primer for amplification. Preferably, the probe comprising a partial nucleic acid sequence of one of the primers used for amplification or the first primer has a length of at least 25 nucleotides, more preferably a length of at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 nucleotides.Preferably, the probe comprising a partial nucleic acid sequence of one of the primers for amplification or the first primer has a length of 25 to 100 nucleotides, more preferably 26 to 100, 26 to 95, 26 to 90, 26 to 85, 26 to 80, 26 to 75, 26 to 70, 26 to 65, 26 to 60, 27 to 100, 27 to 95, 27 to 85, 26 to 80, 26 to 75, 26 to 70, 26 to 65, 26 to 60, 27 to 100, 27 to 95, 27 to to 90, 27 to 85, 27 to 80, 27 to 75, 27 to 70, 27 to 65, 28 to 100, 28 to 95, 28 to 90, 28 to 85, 28 to 80, 28 to 75, 28 to 70, 28 to 65, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 32 to 95, 32 to 90, 32 to 85, 32 to 80, 32 to 75, 32 to 70, 32 to 65, 32 to 60, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70 In some embodiments, the present invention comprises a length of 35 to 65, 38 to 100, 38 to 95, 38 to 90, 38 to 85, 38 to 80, 38 to 75, 38 to 70, 38 to 65, 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, or 40 to 65 nucleotides.

[0099] Preferably, the probe comprising a partial nucleic acid sequence of one of the primers used for amplification or the first primer has a length of 25 to 100 nucleotides, more preferably a length of 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 to 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides.

[0100] In order to prevent the formation of complexes between the probe and the primer, the probe should not contain a sequence that is (substantially) completely complementary to one of the other primers present in the sample. When isolating the nucleic acid amplification product by, for example, DNA purification, no primers should be present, and the probe may then contain a sequence that is completely complementary or partially complementary to one of the other primers present in the sample.

[0101] The present invention further relates to a first composition comprising a dNTP monomer containing a first label and an oligonucleotide probe containing a second label. Preferably, the composition is used for amplification of nucleic acid, preferably wherein the amplification is a recombinase polymerase amplification (RPA). Preferably, the composition comprises a recombinase. The first composition according to the present invention may also comprise the features of the first method of the present invention or the features of the second method of the present invention as much as possible.

[0102] The present invention also relates to a second composition, which comprises a first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and can hybridize with the first strand of the DNA of the target DNA sequence; and an oligonucleotide probe, the oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first label, preferably, wherein the probe can bind to or hybridize with the first strand or the second strand of the DNA of the target DNA sequence, and the second strand is complementary to the first strand. Preferably, the composition is used for amplification of nucleic acids, preferably wherein the amplification is a recombinase polymerase amplification (RPA). Preferably, the composition comprises a recombinase. The second composition according to the present invention can also include the features of the second method of the present invention or the features of the first method of the present invention as much as possible.

[0103] Preferably, the composition according to the invention is nuclease-free or protease-free.Preferably, the composition is nuclease-free and protease-free.

[0104] Methods for performing various suitable nucleic acid amplification reactions to amplify the target DNA sequence according to the present invention are well known in the art. Various reviews have mentioned such methods, such as Zhu et al. (BioTechniques, 2020, vol. 69, no. 4) and et al. (Critical Reviews in Biochemistry and Molecular Biology, 2021, vol.56, issue 6, p543-586). For example, for RPA, WO2010141940A1 and Piepenburg et al. describe suitable methods and reagents. These well-known methods include how and when to contact the sample according to the present invention with primers, dNTPs, enzymes, probes and other reaction components required for amplification. For example, the first primer and the second primer for amplifying the target DNA sequence according to the present invention include a sense primer and an antisense primer, which include a sequence complementary to the target DNA sequence, which is suitable for forming a double-stranded DNA molecule including the target DNA sequence during the amplification reaction. In addition, how to detect various methods of the amplified product according to the present invention after amplification or during amplification, also referred to as real-time detection, such as real-time PCR or real-time RPA, is well known in the art.

[0105] According to the method of the present invention, contacting the sample with a recombinase and / or a probe can be performed before, during or after step b of amplifying the target DNA sequence. Preferably, contacting the sample with a recombinase can be performed before, during or after step b of amplifying the target DNA sequence. Preferably, contacting the sample with a probe can be performed before, during or after step b of amplifying the target DNA sequence. For example, if step b is to be subjected to an RPA reaction, the sample must be contacted with a recombinase before or during step b, but an additional recombinase can be added to the sample so as to contact it after the amplification reaction. After any type of amplification reaction, additional recombinases and / or probes can be added to the sample. If an amplification reaction different from the RPA reaction has been performed, the sample can also be contacted with the recombinase for the first time after step b of amplifying the target DNA sequence.

[0106] Detection of nucleic acid amplification products and thereby of target DNA sequences is qualitative (ie presence or absence) but may alternatively be quantitative (ie amount of product). Preferably, detection of nucleic acid amplification products comprises detection, preferably binding, of a probe.

[0107] "Target DNA sequence" is understood herein to mean a molecule comprising the target DNA sequence, preferably a molecule predominantly comprising DNA, such as a DNA molecule. Preferably, the DNA molecule comprises or consists of double-stranded DNA. A DNA sequence, such as an oligonucleotide or a target DNA sequence comprising a dNTP monomer herein is understood to mean that the dNTP monomer is incorporated into the DNA sequence as a nucleotide.

[0108] "Hybridization" herein is understood to mean the combination of a single-stranded nucleic acid molecule (preferably DNA or RNA) and a complementary single-stranded nucleic acid sequence. Although there is one or more mismatches between the sequence of the single-stranded nucleic acid molecule and the complementary single-stranded nucleic acid sequence, binding can still occur. The binding of the primer or probe according to the present invention preferably occurs via hybridization. Regarding mismatches, it is expected that the probe according to the present invention has the same features as the primer used in the RPA reaction, which is disclosed, for example, in Daher et al. (Molecular and Cellular Probes, 2015, vol. 29, issue 2, p116-121) and Liu et al. (World Journal of Microbiology and Biotechnology, 2019, 35, Article number: 95). Those skilled in the art can design suitable probes for the present invention.

[0109] "Contact" herein should be understood to mean any suitable means for delivering a sample to or exposing it to a compound described herein (e.g., a DNA molecule, dNTP or protein) to allow physical and / or chemical interactions between the sample and the compound. Suitably, the interaction can occur between the target DNA sequence in the sample on the one hand and primers, probes, recombinases and DNA-dependent polymerases on the other hand. In some cases, the term "contact" refers to providing the compound described herein (e.g., suspended in a solution) directly to the sample. Alternatively, in some cases, the term "contact" refers to providing the sample to the compound described herein (e.g., attached to a solid support). The term "contact" may also include mixing the sample with the compound by any means known in the art (e.g., vortexing, pipetting and / or stirring). In some cases, the term "contact" may also include incubating the sample with the compound for a sufficient amount of time, such as to allow the target DNA sequence in the sample to bind to the compound. The contact time can be any length, depending on the binding affinity and / or concentration of the compound and / or molecule containing the target DNA sequence in the sample, the concentration and / or incubation conditions (e.g., temperature) of the detection reagent. In some embodiments, the contact time between sample and compound can be at least about 30 seconds, at least about 1 minute, at least about 3 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 25 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours or longer. Alternatively, for example, when necessary reaction components have been consumed, longer incubation time may not be useful. Those skilled in the art can adjust the contact time accordingly.

[0110] In the first method of the present invention, dNTP monomers containing a first label are incorporated into the nucleic acid amplification product during amplification to become nucleotides and provide a first label for detection, while an oligonucleotide probe containing a second label provides a second label for detection when bound to the amplification product.

[0111] According to the first method of the present invention, detecting the nucleic acid amplification product in step c preferably includes binding one or more first labels of the dNTP monomers of the nucleic acid amplification product to the first binding agent, and binding the second label of the probe bound to the nucleic acid amplification product to the second binding agent, preferably wherein the first label is not bound to the second binding agent, and the second label is not bound to the first binding agent. Preferably, at least two first labels are bound by the first binding agent, preferably each by a separate first binding agent or by at least two first binding agents. Preferably, at least three, four, five, six, seven, eight, nine or ten first labels are bound, preferably each by a separate first binding agent. Preferably, at least three, four, five, six, seven, eight, nine or ten first labels are bound, preferably by at least three, four, five, six, seven, eight, nine or ten first binding agents.

[0112] Preferably, the first binding agent is capable of binding to a first label. Preferably, the second binding agent is capable of binding to a second label. Preferably, the first binding agent specifically binds to the first label. Preferably, the second binding agent specifically binds to the second label. Preferably, the first binding agent is attached to a solid support.

[0113] With respect to a binding agent (e.g., an antibody), "capable of binding" or "binding" herein is understood to mean a binding agent that is capable of binding to a specific marker (e.g., a protein or polysaccharide) under the usual experimental conditions of a biological binding assay. In the context of the present invention, a binding agent that binds or is capable of binding to a specific marker (e.g., biotin or digoxin) forms or undergoes physical association with it in an amount and for a time sufficient to allow detection of the binding agent-marker complex. Preferably, the binding agent is capable of specifically binding to a specific marker. In the context of the present invention, a binding agent (or a specifically bound antibody) that specifically binds to a specific marker (e.g., biotin or digoxin) forms or undergoes physical association with the marker in an amount and for a time sufficient to allow detection of the binding agent-marker complex. "Specifically" or "specific for" means that the binding agent has a higher affinity for a specific marker than for other markers (such as, for example, for other markers contained in the sample). In the context of the present invention, when referring to a binding agent (e.g., an antibody or streptavidin), the term "affinity" refers to the strength with which the binding agent binds to a specific marker or a portion thereof, and is measured by the affinity constant (defined as 1 / KD, where KD is the classically defined dissociation constant) between the antibody and the marker. The measurement of the reaction rate constant can be used to define an equilibrium constant or affinity constant (1 / KD). Therefore, the affinity of a binding agent to its target is negatively correlated with the dissociation constant, i.e., the smaller the KD value, the greater the affinity of the binding agent to its target. For example, a binding agent may have an affinity for a specific marker that is at least about 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, or 10 times or more for other markers in a sample. For example, a binding agent may have an affinity for a specific marker that is at least about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, about 5 times, or about 10 times or more for other markers in a sample. Such degrees of affinity or specificity can be determined by a variety of routine procedures, including competitive binding assays.

[0114] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: 30% to 70% deoxycytidine triphosphate (dCTP) containing a first label, 30% to 70% deoxyadenosine (dATP) containing a first label, 30% to 70% deoxyguanosine (dGTP) containing a first label, 30% to 70% deoxythymidine (dTTP) containing a first label, and / or 30% to 70% deoxyuridine triphosphate (dUTP) containing a first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture contains 30% to 70% dCTP containing a first label and / or 30% to 70% dUTP containing a first label.

[0115] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: 30% to 70% biotin-labeled dCTP, 30% to 70% biotin-labeled dATP, 30% to 70% biotin-labeled dGTP, 30% to 70% biotin-labeled dTTP and / or 30% to 70% biotin-labeled dUTP, each based on the total molar number of dNTPs of its base type, wherein the first label is biotin. Preferably, the mixture contains 30% to 70% biotin-labeled dCTP and / or 30% to 70% biotin-labeled dUTP. The inventors have found that the above ratio of biotin-labeled dNTP to unlabeled dNTP leads to improved detection of amplification products.

[0116] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: about 30% to about 70% deoxycytidine triphosphate (dCTP) containing a first label, about 30% to about 70% deoxyadenosine (dATP) containing a first label, about 30% to about 70% deoxyguanosine (dGTP) containing a first label, about 30% to about 70% deoxythymidine (dTTP) containing a first label, and / or about 30% to about 70% deoxyuridine triphosphate (dUTP) containing a first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture contains about 30% to about 70% dCTP containing a first label and / or about 30% to about 70% dUTP containing a first label.

[0117] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: about 30% to about 70% biotin-labeled dCTP, about 30% to about 70% biotin-labeled dATP, about 30% to about 70% biotin-labeled dGTP, about 30% to about 70% biotin-labeled dTTP and / or about 30% to about 70% biotin-labeled dUTP, each based on the total molar number of dNTPs of its base type, wherein the first label is biotin. Preferably, the mixture contains about 30% to about 70% biotin-labeled dCTP and / or about 30% to about 70% biotin-labeled dUTP. The inventors have found that the above ratio of biotin-labeled dNTP to unlabeled dNTP leads to improved detection of amplification products.

[0118] "Base type" herein is understood to mean any type of nitrogenous base: adenine, guanine, thymine, uracil and cytosine, which consist of their corresponding deoxy(ribo)nucleosides.

[0119] For example, a mixture according to the present invention may include 30% dCTP and 70% biotin-labeled dCTP. In another example, a mixture may include 40% dCTP, 60% biotin-labeled dCTP, 50% dUTP, and 50% biotin-labeled dUTP, totaling up to 100% of base type cytosine and 100% of base type uracil. Therefore, the mixture includes a total of 100% of the corresponding dNTPs, wherein a specific percentage contains the first label, and the remaining percentage does not contain the first label. It should be understood that there is a specific mixture for nucleic acid amplification reactions.

[0120] Preferably, the mixture according to the invention comprises from 30% to about 65%, from about 30% to about 60%, from about 30% to about 55%, from about 30% to about 50%, from about 30% to about 45%, from about 35% to about 70%, from about 35% to about 65%, from about 35% to about 60%, from about 35% to about 55%, from about 35% to about 45%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 40% to about 55%, from about 40% to about 50%, from about 45% to about 70%. , about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60% of the first labeled dCTP, the first labeled dATP, the first labeled dGTP, the first labeled dTTP and / or the first labeled dUTP, preferably, the first labeled dCTP and / or the first labeled dUTP, each based on the total moles of dNTPs of their base types. Preferably, the mixture according to the present invention comprises about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% to about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65% of the first labeled dCTP, the first labeled dATP, the first labeled dGTP, the first labeled dTTP and / or the first labeled dUTP, preferably, the first labeled dCTP and / or the first labeled dUTP, each based on the total molar number of dNTPs of its base type. Preferably, the mixture comprises about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 40% to about 55%, about 40% to about 60%, about 45% to about 55%, or about 45% to about 60% of the dCTP containing the first label, based on the total moles of dCTP. Preferably, the mixture comprises about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 40% to about 60%, about 40% to about 55%, about 45% to about 55%, or about 45% to about 60% of the dUTP containing the first label, based on the total moles of dNTPs thereof. Preferably, the mixture comprises about 35% to about 45% of the dCTP containing the first label and about 55% to about 65% of the dUTP containing the first label, based on the total moles of the dNTPs of their base types.

[0121] Preferably, the mixture according to the invention comprises 30% to 65%, 30% to 60%, 30% to 55%, 30% to 50%, 30% to 45%, 35% to 70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 45%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 45% to 70%, 45% to 65%, 40% to 50%, 45% to 70%, 45% to 65%, 40% to 55%, 40% to 50%, 45% to 70%, 45% to 65%, 40% to 50%. 5% to 60%, 45% to 55%, 50% to 70%, 50% to 65%, or 50% to 60% of the dCTP containing the first label, the dATP containing the first label, the dGTP containing the first label, the dTTP containing the first label and / or the dUTP containing the first label, preferably, the dCTP containing the first label and / or the dUTP containing the first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture according to the present invention comprises 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% to 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of dCTP containing a first label, dATP containing a first label, dGTP containing a first label, dTTP containing a first label and / or dUTP containing a first label, preferably dCTP containing a first label and / or dUTP containing a first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture comprises 35% to 60%, 35% to 55%, 35% to 50%, 40% to 55%, 40% to 60%, 45% to 55% or 45% to 60% of the dCTP containing the first label, based on the total moles of dCTP. Preferably, the mixture comprises 35% to 60%, 35% to 55%, 35% to 50%, 40% to 60%, 40% to 55%, 45% to 55% or 45% to 60% of the dUTP containing the first label, based on the total moles of dNTPs thereof. Preferably, the mixture comprises 35% to 45% of the dCTP containing the first label and 55% to 65% of the dUTP containing the first label, based on the total moles of the dNTPs of their base types.

[0122] Preferably, the mixture according to the present invention comprises from 30% to about 65%, from about 30% to about 60%, from about 30% to about 55%, from about 30% to about 50%, from about 30% to about 45%, from about 35% to about 70%, from about 35% to about 65%, from about 35% to about 60%, from about 35% to about 55%, from about 35% to about 45%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 40% to about 55%, from about 40% to about 50%, from about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 50% to about 70%, about 50% to about 65% or about 50% to about 60% biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably, containing a first labeled dCTP and / or containing a first labeled dUTP, each based on the total moles of dNTPs of its base type. Preferably, the mixture according to the present invention comprises about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% to about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65% of biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably, containing a first labeled dCTP and / or containing a first labeled dUTP, each species based on the total molar number of its base type dNTP. Preferably, the mixture comprises about 50% biotinylated dCTP, about 50% biotinylated dUTP, or about 40% biotinylated dCTP and 60% biotinylated dUTP. Preferably, the mixture comprises about 45% to about 55% biotinylated dCTP, about 45% to about 55% biotinylated dUTP, or about 35% to about 45% biotinylated dCTP and about 55% to about 65% biotinylated dUTP, each based on the total moles of dNTPs of its base type.

[0123] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: up to about 55% of dCTP containing a first label, up to about 55% of dATP containing a first label, up to about 55% of dGTP containing a first label, up to about 55% of dTTP containing a first label, and / or up to about 55% of dUTP containing a first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture comprises up to about 55% of dCTP containing a first label and / or up to about 55% of deoxyuridine dUTP containing a first label. Preferably, the mixture comprises up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10% or up to about 5% of dCTP containing a first label, dATP containing a first label, dGTP containing a first label, dTTP containing a first label and / or dUTP containing a first label, preferably dCTP containing a first label and / or dUTP containing a first label, each based on the total molar number of dNTPs of its base type.

[0124] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: up to about 55% biotinylated dCTP, up to about 55% biotinylated dATP, up to about 55% biotinylated dGTP, up to about 55% biotinylated dTTP and / or up to about 55% biotinylated dUTP, each based on the total molar number of dNTPs of its base type, wherein the first label is biotin. Preferably, the mixture comprises up to about 55% biotinylated dCTP and / or up to about 55% biotinylated dUTP. Preferably, the mixture comprises up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10% or up to about 5% biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably containing the first labeled dCTP and / or dUTP, each based on the total moles of dNTPs of its base type.

[0125] Preferably, the mixture according to the invention comprises 30% to 65%, 30% to 60%, 30% to 55%, 30% to 50%, 30% to 45%, 35% to 70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 45%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 45% to 70%, 45% to 70%. % to 65%, 45% to 60%, 45% to 55%, 50% to 70%, 50% to 65%, or 50% to 60% biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably containing the first labeled dCTP and / or dUTP, each based on the total molar number of dNTPs of its base type. Preferably, the mixture according to the invention comprises 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% to 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65% of biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably containing the first labeled dCTP and / or dUTP, each based on the total molar number of dNTPs of its base type. Preferably, the mixture comprises 45% to 55% biotinylated dCTP, 45% to 55% biotinylated dUTP, or 35% to 45% biotinylated dCTP and 55% to 65% biotinylated dUTP, each based on the total molar number of dNTPs of its base type.

[0126] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: up to 55% dCTP containing the first label, up to 55% dATP containing the first label, up to 55% dGTP containing the first label, up to 55% dTTP containing the first label and / or up to 55% dUTP containing the first label, each based on the total molar number of dNTPs of its base type. Preferably, the mixture contains up to 55% dCTP containing the first label and / or up to 55% deoxyuridine dUTP containing the first label. Preferably, the mixture comprises up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10% or up to 5% of dCTP containing a first label, dATP containing a first label, dGTP containing a first label, dTTP containing a first label and / or dUTP containing a first label, preferably dCTP containing a first label and / or dUTP containing a first label, each based on the total molar number of dNTPs of its base type.

[0127] According to the first method of the present invention, contacting the sample with a dNTP monomer containing a first label preferably includes contacting the sample with a mixture comprising: up to 55% biotin-labeled dCTP, up to 55% biotin-labeled dATP, up to 55% biotin-labeled dGTP, up to 55% biotin-labeled dTTP and / or up to 55% biotin-labeled dUTP, each based on the total molar number of dNTPs of its base type, wherein the first label is biotin. Preferably, the mixture comprises up to 55% biotin-labeled dCTP and / or up to 55% biotin-labeled dUTP. Preferably, the mixture comprises up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10% or up to 5% biotinylated dCTP, biotinylated dATP, biotinylated dGTP, biotinylated dTTP and / or biotinylated dUTP, preferably containing the first labeled dCTP and / or dUTP, each based on the total moles of dNTPs of its base type.

[0128] In the second method of the present invention, one or more dNTPs containing the first label have been incorporated into the oligonucleotide probe, and therefore are also referred to as nucleotides. When the probe is combined with the target DNA sequence, these provide the first label for detection. The second primer contains the second label, and when the primer hybridizes with the target DNA sequence, the second label for detection is provided.

[0129] According to the second method of the present invention, the second primer preferably hybridizes with the first strand of DNA of the target DNA sequence during step b of amplifying the target DNA sequence to produce a nucleic acid amplification product.

[0130] According to the second method of the present invention, the probe preferably hybridizes to the first strand or the second strand of DNA of the target DNA sequence, the second strand being complementary to the first strand.

[0131] According to the second method of the present invention, in step c, detecting the nucleic acid amplification product preferably includes combining one or more first labels of the nucleotides of the probe with a first binding agent, and combining the second label of the second primer bound to the nucleic acid amplification product with a second binding agent, preferably wherein the first label is not bound to the second binding agent, and the second label is not bound to the first binding agent. Preferably, the first binding agent is capable of binding to the first label. Preferably, the second binding agent is capable of binding to the second label. Preferably, the first binding agent specifically binds to the first label. Preferably, the second binding agent specifically binds to the second label. Preferably, the second binding agent is attached to a solid support.

[0132] According to the second method of the present invention, the probe preferably comprises at least two nucleotides containing a first label. Preferably, the probe comprises at least three, four, five, six, seven, eight, nine or ten nucleotides containing a first label.

[0133] According to the second method of the present invention, the probe preferably comprises about 10%, about 20%, about 30% or about 40% to about 60%, about 70%, about 80% or about 90% of nucleotides containing the first label, based on the total number of nucleotides in the probe. According to the second method of the present invention, the probe preferably comprises about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 10% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 90%, about 30% to about 80%, about From about 30% to about 70%, from about 30% to about 60%, from about 30% to about 50%, from about 30% to about 40%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 90%, from about 60% to about 80%, from about 60% to about 70%, from about 70% to about 90%, from about 70% to about 80%, or from about 80% to about 90% of the nucleotides contain the first label.

[0134] According to the second method of the present invention, the probe preferably comprises 10%, 20%, 30% or 40% to 60%, 70%, 80% or 90% of nucleotides containing the first label based on the total number of nucleotides in the probe. According to the second method of the present invention, the probe preferably comprises 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 90%, 30% to 80% , 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 90%, 70% to 80%, or 80% to 90% of the nucleotides containing the first label.

[0135] According to the present invention, amplifying the target DNA sequence to produce a nucleic acid amplification product preferably includes performing a PCR reaction or an isothermal amplification reaction. Preferably, the target DNA sequence is amplified by performing a PCR reaction or an isothermal amplification reaction. Preferably, the isothermal amplification reaction is a recombinase polymerase amplification (RPA) reaction. According to the present invention, the nucleic acid amplification product preferably comprises double-stranded DNA.

[0136] The sample according to the present invention can be contacted with a probe and a recombinase after amplifying the target DNA sequence according to the present invention, and the amplification is preferably carried out by performing a nucleic acid amplification reaction different from the RPA reaction. The sample according to the present invention can be contacted with a recombinase after amplifying the target DNA sequence according to the present invention, and the amplification is preferably carried out by performing a nucleic acid amplification reaction different from the RPA reaction. Therefore, the recombinase promotes the binding of the probe to the target DNA sequence only after nucleic acid amplification. Preferably, after amplifying the target DNA sequence and before detecting the amplified product, an additional step of separating the nucleic acid amplification product and / or removing the polymerase is performed, and then the nucleic acid amplification product is contacted with the probe and the recombinase. The step of separating the amplified product and / or removing the polymerase especially minimizes the potential off-target or de novo amplification product detected after adding the probe and the recombinase. Alternatively, the sample can be contacted with the probe after amplifying the target DNA sequence according to the present invention, and after the sample has been contacted with the recombinase before or during the amplification, and the amplification is preferably carried out by performing a nucleic acid amplification reaction different from the RPA reaction. In addition, according to the present invention, the sample can be contacted with the probe during or after step b of amplifying the target DNA sequence, preferably wherein the sample is not contacted with the probe before step b, and the amplification is preferably carried out by performing a nucleic acid amplification reaction different from the RPA reaction. According to the invention, the sample may be contacted with the probe after step b, and the sample contacted with a further amount of the recombinase, preferably wherein the sample was not contacted with the probe before step b.

[0137] According to the present invention, the probe preferably comprises a polymerase blocking group, preferably a DNA-dependent DNA polymerase blocking group. According to the present invention, the blocking group prevents polymerase extension of a primer or probe, which can also be used as a primer, particularly during RPA. If there is no polymerase or a component necessary for the basic function of the polymerase, a polymerase blocking group on the probe is not required. In addition, when the polymerase is present under conditions where it does not work or has minimal function, a polymerase blocking group is not required on the probe. For example, a single Taq polymerase for a PCR reaction incorporates about 60 nucleotides / s at 70°C, but incorporates about 0.25 nucleotides / s at 22°C. Although research on the use of higher temperature ranges is ongoing in this field, RPA can be performed at 20°C to 50°C, depending on the enzyme used. Therefore, adding a probe after the PCR reaction (optionally in the presence of a recombinase) does not require the probe to include a polymerase blocking group. In addition, the formation of a recombinase-probe-target DNA sequence complex can occur at a temperature where RPA can be performed. According to the present invention, preferably, contacting the recombinase and the probe with the sample (preferably wherein the sample comprises a nucleic acid amplification product) is carried out between about 20° C. and about 50° C., preferably below about 45° C., more preferably below about 40° C., even more preferably below about 35° C., about 30° C. or about 25° C. According to the present invention, preferably, contacting the recombinase and the probe with the sample, preferably wherein the sample comprises a nucleic acid amplification product, is carried out between 20° C. and 50° C., preferably below 45° C., more preferably below 40° C., even more preferably below 35° C., 30° C. or 25° C.

[0138] Various means for blocking different polymerases are known in the art, and can be suitably selected depending on specific determination.For example, modified nucleotides or dNTPs can be used, such as dATP, dCTP, dGTP, dUTP and / or dTTP.In theory, modified nucleotides or dNTPs can be modified at any site of the molecule, but preferably not at the site required for the nucleotides or dNTPs modified to be incorporated into DNA, not at the site required for binding by recombinase, recombinase loading factor or single-stranded binding protein, or not at any other site that hinders or prevents the method according to the present invention from being modified.For example, the dNTP containing the first mark can not be modified to make the 5' phosphate and / or 3' hydroxyl of the deoxyribose of the dNTP be replaced by the first mark.Those skilled in the art can suitably design the nucleotides at the dNTP and modified sites for the method of the present invention.Preferably, the probe according to the present invention comprises the nucleotides containing the polymerase blocking group, and preferably wherein the nucleotides are modified. Preferably, the polymerase blocking group according to the present invention comprises a hydrocarbon spacer, an inverted deoxynucleotide, a phosphorylation, a primary amine or a dideoxynucleotide, preferably wherein the hydrocarbon spacer comprises 3 to 12 carbon atoms, preferably 3 to 9, 3 to 6, 6 to 12 or 6 to 9 carbon atoms, more preferably wherein the hydrocarbon spacer comprises 3, 6 or 12 carbon atoms. Preferably, the polymerase blocking group according to the present invention or a nucleotide comprising a polymerase blocking group is located at the 3' end of the probe, wherein the 3' end is 0 to 10 nucleotides of the 3' terminal nucleotide of the probe, inclusive of the 3' terminal nucleotide. Preferably, the 3' end is 0 to 7, 0 to 5, 0 to 3, 3 to 10, 5 to 10, 7 to 10, 3 to 5 or 3 to 7 nucleotides of the 3' terminal nucleotide of the probe, inclusive of the 3' terminal nucleotide. Preferably, the polymerase blocking group or the nucleotide comprising a polymerase blocking group according to the present invention is located at or is the 3' terminal nucleotide or nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 counting from the 3' terminal nucleotide of the probe, respectively.

[0139] Various means for labeling nucleotides, dNTPs, primers or probes are known in the art and can be suitably selected depending on specific determination. For example, fluorescent compounds, enzymes, high affinity proteins or radioactive elements can be attached by various means. Haptens have been widely studied as markers for nucleotides or dNTPs. Marking according to the present invention preferably comprises haptens. Marking according to the present invention preferably comprises covalently attached markers. Preferably, primers or probes containing the first, second, third or fourth markers comprise nucleotides containing the corresponding first, second, third or fourth markers. Preferably, marking, preferably the second, third or fourth marker comprises fluorescein or its derivatives, digoxin, Texas red, dansyl, Cascade blue or dinitrophenol (DNP). Many fluorescein derivatives are known, but preferred fluorescein derivatives include fluorescein amides such as 6-FAM or fluorescein isothiocyanate (FITC). Preferably, marking, preferably the second, third or fourth marker comprises a fluorescent compound or its derivatives, a fluorogenic compound or its derivatives, or a steroid or its derivatives. Preferably, the label, preferably the second, third or fourth label comprises a fluorescent compound containing an aromatic group or a fluorogenic compound containing an aromatic group.The first label according to the invention preferably comprises biotin.

[0140] According to the present invention, the second, third or fourth marker or the nucleotide containing the second, third or fourth marker is preferably located at the 5' end of the probe, wherein the 5' end is 0 to 10 nucleotides of the 5' terminal nucleotide of the probe, including the terminal nucleotide. Preferably, the 5' end is 0 to 7, 0 to 5, 0 to 3, 3 to 10, 5 to 10, 7 to 10, 3 to 5 or 3 to 7 nucleotides of the 5' terminal nucleotide of the probe, including the 5' terminal nucleotide. Preferably, the second, third or fourth marker or the nucleotide containing the second, third or fourth marker according to the present invention is located at or is the 5' terminal nucleotide or nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 counted from the 5' terminal nucleotide of the probe, respectively. According to the present invention, the second, third or fourth marker or the nucleotide containing the second, third or fourth marker is preferably located at the 3' end of the probe, wherein the 3' end is 0 to 10 nucleotides of the 3' terminal nucleotide of the probe, including the terminal nucleotide. Preferably, the 3' end is 0 to 7, 0 to 5, 0 to 3, 3 to 10, 5 to 10, 7 to 10, 3 to 5 or 3 to 7 nucleotides of the 3' terminal nucleotide of the probe, including the 3' terminal nucleotide. Preferably, the second, third or fourth marker or the nucleotide containing the second, third or fourth marker according to the present invention is located at or is the 3' terminal nucleotide or nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 counted from the 3' terminal nucleotide of the probe, respectively. According to the present invention, the second, third or fourth marker or the nucleotide containing the second, third or fourth marker is preferably in the middle of the probe, wherein the middle is 0 to 10 nucleotides of the central nucleotide of the probe, including the central nucleotide. Preferably, the middle is 0 to 7, 0 to 5, 0 to 3, 3 to 10, 5 to 10, 7 to 10, 3 to 5 or 3 to 7 nucleotides of the central nucleotide of the probe, including the central nucleotide. Preferably, the second, third or fourth label or the nucleotide containing the second, third or fourth label according to the present invention is located at or is the central nucleotide of the probe or nucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 counted from the central nucleotide of the probe.

[0141] According to the present invention, the second label is preferably a label different from the first label. Preferably, the first label according to the present invention comprises or is biotin or a derivative thereof, and the second label is not or does not comprise biotin or a derivative thereof. Preferably, the first label according to the present invention comprises or is biotin, and the second label is not or does not comprise biotin. Preferably, the nucleotide or dNTP monomer containing the first label comprises biotin-labeled dCTP, biotin-labeled dUTP, biotin-labeled deoxyguanosine triphosphate (dGTP), biotin-labeled deoxythymidine (dTTP) and / or biotin-labeled deoxyadenosine triphosphate (dATP), more preferably biotin-labeled dCTP and / or biotin-labeled dUTP.

[0142] According to the present invention, the nucleotide or dNTP monomer containing the first label is preferably a biotin-labeled nucleotide or a biotin-labeled dNTP monomer, preferably wherein biotin is preferably covalently linked to the nucleotide or dNTP via a linker of 11 to 18 atoms. Preferably, the linker is 11 to 16, 11 to 14, 14 to 18, 14 to 16 or 16 to 18 atoms, more preferably the linker is 11, 14, 16 or 18 atoms, and most preferably the linker is 16 atoms. Preferably, the biotin-labeled dNTP monomer comprises biotin-labeled dCTP, biotin-labeled dATP, biotin-labeled dGTP, biotin-labeled dTTP and / or biotin-labeled dUTP, more preferably biotin-labeled dCTP and / or biotin-labeled dUTP, even more preferably biotin-16-dCTP and / or biotin-16-dUTP. Preferably, the biotin-labeled nucleotides comprise biotin-labeled cytosine nucleotides, biotin-labeled adenine nucleotides, biotin-labeled guanine nucleotides, biotin-labeled thymidine nucleotides and / or biotin-labeled uracil nucleotides, more preferably biotin-labeled cytosine nucleotides and / or biotin-labeled uracil nucleotides.

[0143] According to the present invention, based on the total number of dNTP monomers of the probe, the probe preferably comprises about 30% to about 70%, preferably about 40% to about 50% dCTP and / or dGTP monomers, optionally wherein the monomers contain a first label. Preferably, based on the total number of dNTP monomers of the probe, the probe comprises about 30% to about 60%, about 30% to about 50%, about 40% to about 70% or about 40% to about 60% dCTP and / or dGTP monomers, optionally wherein the monomers contain a first label. According to the present invention, based on the total number of nucleotides of the probe, the probe preferably comprises about 30% to about 70%, preferably about 40% to about 50% cytosine and / or guanine nucleotides, optionally wherein the nucleotides contain a first label. Preferably, based on the total number of nucleotides of the probe, the probe comprises about 30% to about 60%, about 30% to about 50%, about 40% to about 70% or about 40% to about 60% cytosine and / or guanine nucleotides, optionally wherein the nucleotides contain a first label.

[0144] According to the present invention, based on the total number of dNTP monomers of the probe, the probe preferably comprises 30% to 70%, preferably 40% to 50% dCTP and / or dGTP monomers, optionally wherein the monomers contain a first label. Preferably, based on the total number of dNTP monomers of the probe, the probe comprises 30% to 60%, 30% to 50%, 40% to 70% or 40% to 60% dCTP and / or dGTP monomers, optionally wherein the monomers contain a first label. According to the present invention, based on the total number of nucleotides of the probe, the probe preferably comprises 30% to 70%, preferably 40% to 50% cytosine and / or guanine nucleotides, optionally wherein the nucleotides contain a first label. Preferably, based on the total number of nucleotides of the probe, the probe comprises 30% to 60%, 30% to 50%, 40% to 70% or 40% to 60% cytosine and / or guanine nucleotides, optionally wherein the nucleotides contain a first label.

[0145] In US20110059506A1, it has been suggested that during RPA, gradually larger oligonucleotides can reduce the rate of invasion / extension, although it may be necessary to extend the length of the oligonucleotide to accommodate the search for duplex regions in the 3' or 5' region of the oligonucleotide. Therefore, according to the present invention, the probe or primer preferably has a length of at least 25 nucleotides, more preferably at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55 or at least 60 nucleotides. Preferably, the probe or primer has a length of 25 to 100 nucleotides, more preferably a length of 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 to 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 80, 90, or 100 nucleotides, even more preferably a length of 30, 35, or 40 to 45, 50, 55, or 60 nucleotides. Preferably, the probe or primer has a length of 25 to 100 nucleotides, more preferably 26 to 100, 26 to 95, 26 to 90, 26 to 85, 26 to 80, 26 to 75, 26 to 70, 26 to 65, 26 to 60, 27 to 100, 27 to 95, 27 to 90, 27 to 85, 27 to 80, 27 to 75, 27 to 70, 27 to 65, 28 to 100, 28 to 95, 28 to 90, 28 to 85, 28 to 80, 28 to 75, 28 to 70, 28 to 65, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75 , 30 to 70, 30 to 65, 32 to 95, 32 to 90, 32 to 85, 32 to 80, 32 to 75, 32 to 70, 32 to 65, 32 to 60, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 In some embodiments, the present invention relates to a polypeptide having a length of at least 38, 38 and 100 nucleotides, at least 38 and 95 nucleotides, at least 38 and 90 nucleotides, at least 38 and 85 nucleotides, at least 38 and 80 nucleotides, at least 38 and 75 nucleotides, at least 38 and 70 nucleotides, or at least 40 nucleotides in length.

[0146] The DNA-dependent DNA polymerase according to the present invention is preferably a strand-displacement DNA-dependent DNA polymerase so as to be able to perform an RPA reaction.

[0147] Recombinase plays a central role in homologous recombination of various organisms. Such recombinases have been described in archaea, bacteria, eukaryotes and viruses. The recombinase according to the present invention is preferably a recombinase that promotes homologous recombination. Preferably, the recombinase according to the present invention is a recombinase that can target an oligonucleotide (such as a primer or an oligonucleotide probe) to a target DNA sequence (preferably in double-stranded DNA). Preferably, the recombinase can pair an oligonucleotide (such as a primer or an oligonucleotide probe) with a complementary DNA sequence (preferably in double-stranded DNA). Preferably, the recombinase according to the present invention is a recombinase selected from the group consisting of: RecA, DrRecA, Rad51, RadA or UvsX recombinase or its homologue. The recombinase RecA family is encoded by several organisms. Preferably, the recombinase is the RecA of Escherichia coli or Deinococcus radiodurans. Preferably, the recombinase is the UvsX recombinase of bacteriophage T4. Preferably, the recombinase is Rad51 and its cofactor. Preferably, the recombinase is DrRecA of Deinococcus radiodurans. These recombinases promote the formation of the D ring. Preferably, the recombinase does not use or require nucleoside triphosphates such as ATP or GTP to form or maintain the D ring. Preferably, the recombinase does not use or require analogs of nucleoside triphosphates such as ATPγS to form or maintain the D ring.

[0148] The method according to the present invention is preferably a one-pot reaction. The one-pot reaction simplifies the method and reduces the (cross) contamination of the sample. Preferably, the one-pot reaction comprises all steps of the method being carried out simultaneously. Preferably, the one-pot reaction comprises carrying out the method in one container. Preferably, the one-pot reaction does not include purification or separation of the compound.

[0149] According to the present invention, the method preferably includes contacting the sample with a component for regulating the combination of a recombinase or labeled probe with a nucleic acid amplification product (preferably comprising a sample of a nucleic acid amplification product or a separated nucleic acid amplification product). As disclosed above, the combination of a labeled probe or a recombinase labeled probe complex with a target DNA sequence of a nucleic acid amplification product promotes the subsequent detection of the nucleic acid amplification product. Therefore, it is preferred to regulate this combination to maintain or increase the combination. Preferably, the component for regulating the combination of a recombinase or labeled probe with a nucleic acid amplification product includes ATP analogs, RecO, RecT, RecF, UvsY, double-stranded DNA destabilizer, triple-stranded DNA stabilizer, single-stranded DNA binding protein or any combination thereof.

[0150] It is speculated that the binding of the recombinase to the single-stranded DNA can be stabilized by using a non-hydrolyzable or hydrolysis-resistant ATP analogue, because in the presence of the non-hydrolyzable ATP analogue 5'-γ-thioadenosine triphosphate (ATP-γ-S), the hydrolysis of ATP-γ-S by the recombinase and the subsequent dissociation of the binding of the recombinase to the single-stranded DNA are inhibited (see US20110059506A1). It is proposed that the stability of the RecA / ATP-γ-S / DNA complex may be helpful for targeting the recombinase, but may also be detrimental and impractical for DNA amplification.

[0151] According to the present invention, the method preferably also includes adding an ATP analogue before, during or after amplifying the target DNA sequence, preferably after amplification. Preferably, amplifying the target DNA sequence includes performing an RPA reaction and adding an ATP analogue during or after the RPA reaction. Adding an ATP analogue before or during amplifying the target DNA sequence preferably includes contacting the sample with an ATP analogue. Adding an ATP analogue after amplifying the target DNA sequence preferably includes contacting the nucleic acid amplification product with an ATP analogue, optionally after separating the nucleic acid amplification product. The term "adding" herein should be understood to mean contacting the sample with it.

[0152] Preferably, the ATP analogue according to the present invention is a non-hydrolyzable or hydrolysis-resistant ATP analogue. Preferably, the ATP analogue is selected from the group consisting of: AMPPNP adenosyl imide diphosphate (AMPPNP), adenosyl methylene diphosphate (AMPPCP), α,β-methylene triphosphate (AMPCPP), α-thio-5-triphosphate (ATPαS) and adenosine 5'-γ-thio triphosphate (ATP-γ-S). Preferably, the ATP analogue is ATP-γ-S.

[0153] It has been found that adding ATP analogs together with ATP is advantageous for the method of the present invention. According to the present invention, the method preferably also includes adding ATP analogs and ATP before, during or after amplifying the target DNA sequence, preferably after amplification. Preferably, amplifying the target DNA sequence includes performing an RPA reaction and adding ATP analogs and ATP during or after the RPA reaction. Adding ATP analogs and ATP before or during amplifying the target DNA sequence preferably includes contacting the sample with ATP analogs and ATP. Adding ATP analogs and ATP after amplifying the target DNA sequence preferably includes contacting the nucleic acid amplification product with ATP analogs and ATP, optionally after separating the nucleic acid amplification product. Preferably, adding ATP analogs and ATP includes contacting the sample with ATP analogs and ATP in a molar ratio of 0.5 to 2: 5 to 15, preferably about 1: 10. Preferably, adding ATP analogs and ATP includes contacting the sample with ATP analogs and ATP in a molar ratio of 0.5 to 1.5: 5 to 15, 1 to 2: 5 to 15, 0.5 to 1.5: 7 to 13, or 1 to 2: 7 to 13. Preferably, adding the ATP analog and ATP comprises contacting the sample with at least about 2 times the molar amount of the ATP analog and the ATP analog, preferably at least about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times the molar amount of the ATP analog. Preferably, adding the ATP analog and ATP comprises contacting the sample with at least about 2 times the molar amount of the ATP analog and the ATP analog, preferably at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times the molar amount of the ATP analog.

[0154] Alternatively or additionally, ATP can be added by using an ATP regeneration system, for example, by using creatine phosphate and creatine kinase, wherein creatine kinase phosphorylates adenosine monophosphate (AMP) or adenosine diphosphate (ADP) to ATP in the presence of creatine phosphate, or as disclosed in EP3249056B1. The ATP regeneration system generally includes the use of an enzymatic reaction that continuously produces ATP from ADP or AMP, preferably ADP and a secondary phosphate donor. According to the present invention, the method preferably also includes adding an ATP analog and an ATP regeneration system before, during or after amplifying the target DNA sequence, preferably after amplification. Preferably, amplifying the target DNA sequence includes performing an RPA reaction and adding an ATP analog and an ATP regeneration system during or after the RPA reaction. Adding an ATP analog and an ATP regeneration system before or during amplification of the target DNA sequence preferably includes contacting the sample with an ATP analog and an ATP regeneration system. Adding an ATP analog and an ATP regeneration system after amplifying the target DNA sequence preferably includes contacting the nucleic acid amplification product with an ATP analog and an ATP regeneration system, optionally after separating the nucleic acid amplification product. Preferably, amplifying the target DNA sequence comprises performing an RPA reaction.Preferably, the ATP regeneration system comprises creatine phosphate and creatine kinase, acetate kinase and acetyl phosphate, pyruvate kinase and phosphoenolpyruvate and / or phospho-phenyl-pyruvate, or polyphosphate kinase and polyphosphate, more preferably creatine phosphate and creatine kinase.

[0155] In US20110059506A1, it is proposed that the RecA-ssDNA complex can be stabilized by RecO, RecT and / or RecF proteins, and the UvsX-ssDNA complex can be stabilized by T4UvsY proteins. Therefore, according to the present invention, the sample is preferably also contacted with RecO, RecT, RecF or UvsY. Preferably, when the sample is contacted with the UvsX recombinase, the sample is contacted with UvsY. Preferably, when the sample is contacted with the RecA recombinase, the sample is contacted with RecO, RecT or RecF.

[0156] According to the present invention, the method preferably also includes contacting a sample, preferably a sample containing a nucleic acid amplification product or a nucleic acid amplification product separated by, for example, DNA purification, with a double-stranded DNA destabilizer and / or a triple-stranded DNA stabilizer. Preferably, a triple-stranded DNA stabilizer is an agent capable of stabilizing a complex comprising a recombinase / single-stranded DNA complex and a double-stranded DNA. Preferably, a triple-stranded DNA stabilizer is an agent capable of stabilizing a D-ring, preferably a D-ring produced by a recombinase. Some double-stranded DNA destabilizers can (indirectly) act as triple-stranded DNA stabilizers, and vice versa. Agents that destabilize double-stranded DNA can be used to promote the binding of a probe according to the present invention to a target DNA sequence, thereby indirectly increasing the stability of the D-ring. Examples of double-stranded DNA destabilizers include, but are not limited to, polyethylene glycol (PEG), trehalose, ionic liquids, betaine, proline, adenovirus DNA binding protein, calf thymus helical destabilizing protein, and ribonuclease ( and McCalla, Sensors and Actuators Reports, Volume 3, November 2021, 100033). Ribonucleases can destabilize RNA-DNA duplexes. Preferably, PEG has an average molecular weight of 1450, 6000 or 15000 to 20000. In the present application, the use of ionic liquids surprisingly enables improved detection of nucleic acid amplification products according to the present invention, see Fig. 9 F. This seems to be particularly true when using a probe comprising a DNA sequence complementary to the middle region of the target DNA sequence, when a paranemic joint is formed. An example of a triple-stranded DNA stabilizer is potassium, preferably KCl (Teng et al., Molecules, 2020, 25(2), 387). The triple-stranded DNA stabilizer comprising potassium may also comprise PEG, preferably PEG 200. Additional double-stranded DNA destabilizers and triple-stranded DNA stabilizers are known in the art and may be suitably selected for use according to the present invention.

[0157] According to the present invention, the sample is preferably also contacted with a single-stranded DNA binding protein. Preferably, the single-stranded DNA binding protein comprises gp32 of bacteriophage T4 or single-stranded binding protein SSB of Escherichia coli. Preferably, when the sample is contacted with UvsX recombinase, the sample is contacted with gp32. Preferably, when the sample is contacted with RecA recombinase, the sample is contacted with SSB. Single-stranded DNA binding proteins help stabilize the displaced DNA strands, which promote the formation of the D-loop.

[0158] The presence or absence of a target ribonucleic acid (RNA) sequence can also be determined by the present invention. To achieve this, first the target RNA sequence is converted into DNA, for example, into complementary DNA (cDNA), via a process known as reverse transcription, which is a process well known in the art. Therefore, a target DNA sequence is produced from the target RNA sequence. According to the present invention, the method may also include contacting the sample with an RNA-dependent DNA polymerase before or during step a or b to produce a target DNA sequence.

[0159] According to the present invention, the nucleic acid sequence complementary to the target DNA sequence in the probe or the nucleic acid sequence hybridized to the probe in the target DNA sequence preferably does not overlap with the nucleic acid sequence hybridized to the first primer or the second primer. According to the present invention, the nucleic acid sequence complementary to the target DNA sequence in the probe or the nucleic acid sequence hybridized to the probe preferably overlaps with the nucleic acid sequence partially hybridized to the first primer or the second primer, preferably wherein the partial overlap is 1 to 10, 1 to 7, 1 to 5, 1 to 3, 3 to 7, 3 to 5 or 5 to 7 nucleotides. The degree of partial overlap of primers and probe sequences depends on the length of the probe and the needs of potential mispairing discrimination, and can be suitably selected by those skilled in the art.

[0160] According to the present invention, contacting the sample preferably comprises contacting the sample with a smaller or equal amount of the probe than the amount of the first primer and / or the second primer on a molar basis, preferably wherein the molar ratio of the first primer and the second primer to the probe is 1:1 to 100:1, 1:1 to 75:1, 1:1 to 25:1, 10:1 to 100:1, 25:1 to 100:1, 10:1 to 75:1, 10:1 to 25:1, or 25:1 to 75:1. Preferably, contacting the sample comprises contacting the sample with primers and probes, the molar ratio of primers to probes being 1:1 to 100:1, 1:1 to 75:1, 1:1 to 25:1, 10:1 to 100:1, 25:1 to 100:1, 10:1 to 75:1, 10:1 to 25:1, or 25:1 to 75:1.

[0161] The first binding agent according to the present invention preferably comprises a binding agent capable of binding to biotin, preferably avidin, streptavidin, neutravidin, an anti-biotin antibody or a binding agent specific for biotin. The second binding agent according to the present invention preferably comprises an antibody. According to the present invention, the first binding agent preferably comprises a binding agent capable of binding to biotin, preferably avidin, streptavidin, neutravidin, an anti-biotin antibody or a binding agent specific for biotin, and the second binding agent comprises a binding agent or antibody that cannot bind to biotin or does not specifically bind to biotin.

[0162] Various means of detecting labeled compounds are known in the art. Detection according to the present invention is preferably achieved via visual readout, preferably without the aid of advanced equipment. According to the present invention, detecting nucleic acid amplification products preferably includes the use of lateral flow tests, lateral flow devices, enzyme-linked immunosorbent assays (ELISA), magnetic immunoassays or flocculation assays. According to the present invention, the first binding agent preferably includes nanoparticles, colored (nano)particles, gold (nano)particles, latex, fluorescent compounds, luminescent compounds or magnetic compounds. The increase in the local concentration of the first label after amplification can facilitate (visual) detection by the above-mentioned first binding agent. Real-time detection during amplification can also be performed using the method of the present invention. Various methods of real-time detection during nucleic acid amplification can be achieved by combining known methods for real-time detection of real-time PCR or real-time RPA. For example, a non-specific fluorescent compound embedded in any double-stranded DNA can be contacted with the sample. Optionally, the probe according to the present invention may include a fluorescent label that can only be detected after the probe binds to its complementary sequence. For example, the probe can be designed to exo kit compatible, wherein the exonuclease cleaves specific residues in the hybridized probe, which destroys the quenching effect of the attached quenching compound on the attached fluorophore. According to the present invention, detecting the nucleic acid amplification product is preferably performed during the amplification of the target DNA sequence, preferably wherein the probe, the first label or the second label comprises a fluorescent compound, more preferably the probe.

[0163] The present invention can also be used for multiplex assays, in which multiple target DNA sequences can be amplified and detected simultaneously. According to the first method of the present invention, the method preferably also includes contacting the sample with: a third primer and a fourth primer for amplifying a second target DNA sequence; and a second oligonucleotide probe comprising a nucleic acid sequence complementary to the second target DNA sequence and a third label. According to the first method of the present invention, detecting the nucleic acid amplification product in step c preferably also includes combining the third label of the second probe bound to the nucleic acid amplification product with a third binding agent, preferably wherein the first label and the third label are not bound to the second binding agent, the second label and the third label are not bound to the first binding agent, and the first label and the second label are not bound to the third binding agent. Preferably, the first binding agent is capable of binding to the first label, the second binding agent is capable of binding to the second label, and the third binding agent is capable of binding to the third label. Preferably, the first binding agent is specific to the first label, the second binding agent is specific to the second label, and the third binding agent is specific to the third label. Similarly, additional different primer sets and oligonucleotide probes with different labels can be used to detect additional different target DNA sequences, which can be detected by additional binding agents that can bind or are preferably specific to the respective different labels, for example, the fifth and sixth primers for amplifying a third target DNA sequence, and a third oligonucleotide probe comprising a nucleic acid sequence complementary to the third target DNA sequence and a fourth label; the seventh and eighth primers for amplifying a fourth target DNA sequence, and a fourth oligonucleotide probe comprising a nucleic acid sequence complementary to the fourth target DNA sequence and a fifth label, and so on.

[0164] According to the second method of the present invention, the method preferably further comprises contacting the sample with: a third primer and a fourth primer for amplifying a second target DNA sequence, wherein the fourth primer contains a third label and hybridizes with the first strand of the DNA of the second target DNA sequence, and a second oligonucleotide probe, wherein the second oligonucleotide probe comprises a nucleic acid sequence complementary to the second target DNA sequence and one or more nucleotides containing the first label, preferably wherein the probe hybridizes with the first strand or the second strand of the DNA of the second target DNA sequence, and the second strand is complementary to the first strand. According to the second method of the present invention, in step c, detecting the nucleic acid amplification product preferably further comprises binding the third label of the fourth primer bound to the nucleic acid amplification product to a third binding agent, preferably wherein the first label and the third label are not bound to the second binding agent, the second label and the third label are not bound to the first binding agent, and the first label and the second label are not bound to the third binding agent. Preferably, the first binding agent is capable of binding to the first label, the second binding agent is capable of binding to the second label, and the third binding agent is capable of binding to the third label. Preferably, the first binding agent is specific to the first label, the second binding agent is specific to the second label, and the third binding agent is specific to the third label. Similarly, additional different primer sets (one of the primers has a different label) and oligonucleotide probes targeting different DNA sequences can be used to detect additional different target DNA sequences, which can be detected by additional binding agents that can bind to or are preferably specific for the corresponding different labels, for example, the fifth and sixth primers for amplifying a third target DNA sequence, wherein the sixth primer contains a third label and hybridizes with the first strand of DNA of the third target DNA sequence, and a third oligonucleotide probe, the third oligonucleotide probe comprises a nucleic acid sequence complementary to the third target DNA sequence and one or more nucleotides containing the first label, wherein the probe preferably hybridizes with the first strand or the second strand of DNA of the third target DNA sequence, the second strand being complementary to the first strand; the seventh and eighth primers for amplifying a fourth target DNA sequence, wherein the eighth primer contains a fourth label and hybridizes with the first strand of DNA of the second target DNA sequence, and a fourth oligonucleotide probe, the fourth oligonucleotide probe comprises a nucleic acid sequence complementary to the fourth target DNA sequence and one or more nucleotides containing the first label, wherein the probe preferably hybridizes with the first strand or the second strand of DNA of the fourth target DNA sequence, the second strand being complementary to the first strand, and so on.

[0165] Detection of the multiplex method of the present invention can be carried out by various means known in the art, for example by capturing each of the second and additional labels with corresponding (immobilized) binding agents separated by time or position, after which each target DNA sequence is separated and can be detected individually via the first binding agent.

[0166] The preferred method for detecting the nucleic acid amplification product according to the present invention includes the use of a lateral flow device, also referred to as a lateral flow test (Sajid et al., Journal of Saudi Chemical Society. 2015Nov; Vol 19, Issue 6, p689-705). The lateral flow device is a simple and affordable point-of-care test device to detect the presence of a target substance in a (liquid) sample without the need for specialized and expensive equipment. A typical lateral flow device includes a strip containing a sample application zone, a reagent zone, a test zone, and a terminal zone. The liquid sample to be analyzed is applied to the sample application zone and passes through different zones of the lateral flow band toward the terminal zone. The reagent zone typically includes a reagent capable of subsequently detecting the target substance in the test zone. The test zone typically contains a fixed binding agent that interacts with the target substance, thereby typically indicating the presence of the target substance as a visual line.

[0167] According to the present invention, detecting the nucleic acid amplification product preferably comprises:

[0168] a. providing a lateral flow device comprising a first binding agent capable of binding a first marker and a second binding agent capable of binding a second marker, preferably wherein the second binding agent is immobilized, preferably immobilized on the lateral flow device;

[0169] b. applying the nucleic acid amplification product to a lateral flow test strip of a lateral flow device; and

[0170] c. Detect product (if present) on the test zone of the lateral flow strip.

[0171] Preferably, before step b of applying the nucleic acid amplification product to the lateral flow test strip of the lateral flow device, a step of diluting the nucleic acid amplification product is performed, preferably using a buffer having a pH of about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8 or about 6.9 to about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8, preferably about 7; and then step b of applying the diluted nucleic acid amplification product to the lateral flow test strip of the lateral flow device. Preferably, prior to step b of applying the nucleic acid amplification product to a lateral flow test strip of a lateral flow device, a step of diluting the nucleic acid amplification product is performed, preferably using a 2-well plate having a viscosity of about 6 to about 8, about 6 to about 7.7, about 6 to about 7.5, about 6 to about 7.3, about 6 to about 7.1, about 6.3 to about 8, about 6.3 to about 7.7, about 6.3 to about 7.5, about 6.3 to about 7.3, about 6.3 to about 7.1, about 6.5 to about 8, about 6.5 to about 7.7, about 6.5 to about 7.5, about 6.5 to about 7.3, about 6.5 to about 7.1, about 6.7 to about 8, about 6.7 to about 7.7, about 6.7 to about 7.5, about 6.7 to about 7.3, about 6.7 to about 7.1, about 6.9 to about 8, about 6.9 to about 7.7, about 6.9 to about 7.5, about 6.9 to about 7.3, about 6.9 to about 7.1, or a buffer with a pH of about 7; and followed by step b of applying the diluted nucleic acid amplification product to a lateral flow test strip of a lateral flow device.

[0172] Preferably, before step b of applying the nucleic acid amplification product to the lateral flow test strip of the lateral flow device, a step of diluting the nucleic acid amplification product is performed, preferably using a buffer having a pH of 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9 to 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, preferably about 7; and followed by step b of applying the diluted nucleic acid amplification product to the lateral flow test strip of the lateral flow device. Preferably, before step b of applying the nucleic acid amplification product to a lateral flow test strip of the lateral flow device, a step of diluting the nucleic acid amplification product is performed, preferably using a buffer having a pH of 6 to 8, 6 to 7.7, 6 to 7.5, 6 to 7.3, 6 to 7.1, 6.3 to 8, 6.3 to 7.7, 6.3 to 7.5, 6.3 to 7.3, 6.3 to 7.1, 6.5 to 8, 6.5 to 7.7, 6.5 to 7.5, 6.5 to 7.3, 6.5 to 7.1, 6.7 to 8, 6.7 to 7.7, 6.7 to 7.5, 6.7 to 7.3, 6.7 to 7.1, 6.9 to 8, 6.9 to 7.7, 6.9 to 7.5, 6.9 to 7.3, 6.9 to 7.1, or about 7; and followed by step b of applying the diluted nucleic acid amplification product to a lateral flow test strip of the lateral flow device.

[0173] Preferably, the buffer comprises a non-ionic detergent, preferably a non-ionic detergent that does not substantially denature or aggregate the protein. Preferably, the buffer comprises a non-denaturing non-ionic detergent. Various non-denaturing non-ionic detergents are known in the art and can be suitably selected, examples being Tween-20, Tween-80, digitonin, n-dodecyl-β-D-maltoside, Triton X-100, Triton X-114, Nonidet P-40 (NP-40), CA-630. Optionally, the buffer preferably contains a non-denaturing ionic detergent such as bile acid.

[0174] It was found that diluting the nucleic acid amplification product improved the detection of the product. Excess nucleic acid amplification product and / or crowding agents in the RPA reaction may interfere with the generation of a detectable signal, for example by reducing the wicking rate of the sample pad (i.e., sample application zone) and / or the capillary flow in the lateral flow assay, by reducing the amount of binding agent per amplified target DNA sequence, and / or by binding to the first binding agent without binding to the immobilized second binding agent, thereby interfering with the generation of a detectable signal, resulting in a low signal.

[0175] Another preferred method for detecting a nucleic acid amplification product according to the present invention includes flocculation. US2017029881A1 discloses the detection of amplified nucleic acids by visual inspection after flocculation. In this article, the complex is formed by flocculation of nucleic acids and particles capable of binding nucleic acids. US2017029881A1 defines flocculation as the reversible non-covalent aggregation of colloids, which then enables visual inspection of aggregates. Due to the high concentration of the first marker present in the amplified product, the present invention enables visual detection of the nucleic acid amplification product via flocculation, which can then cause a high local concentration of the first binding agent that binds the first marker, thereby generating a visual signal.

[0176] According to the present invention, detecting the nucleic acid amplification product preferably comprises combining the nucleic acid amplification product and a first binding agent, wherein the product and the first binding agent are capable of forming a visually detectable complex, wherein the complex is formed by flocculation, preferably by flocculation of the first binding agent, more preferably by flocculation of the nucleic acid amplification product on the first binding agent. Preferably, the method also comprises that the presence or relative amount of the nucleic acid amplification product is determined by the presence or increased flocculation compared to the flocculation observed in the absence or in the presence of a lower amount or concentration of the nucleic acid amplification product. Preferably, the first binding agent comprises avidin, streptavidin, neutravidin, antibiotin antibody or a binding agent specific for biotin bound to one or more colored, silver or gold nanoparticles (preferably gold nanoparticles). Preferably, the nanoparticles have a diameter of 4nm or 5nm to 50nm or 55nm. Preferably, the nanoparticles have a silica shell with a diameter of 2nm to 4nm. Preferably, the nanoparticles comprise a polyethylene glycol polymer.

[0177] Preferably, the method further comprises diluting the complex using a buffer having a pH of about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8 or about 6.9 to about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8, preferably about 7. Preferably, the method further comprises diluting the complex with a buffer having a pH of about 6 to about 8, about 6 to about 7.7, about 6 to about 7.5, about 6 to about 7.3, about 6 to about 7.1, about 6.3 to about 8, about 6.3 to about 7.7, about 6.3 to about 7.5, about 6.3 to about 7.3, about 6.3 to about 7.1, about 6.5 to about 8, about 6.5 to about 7.7, about 6.5 to about 7.5, about 6.5 to about 7.3, about 6.5 to about 7.1, about 6.7 to about 8, about 6.7 to about 7.7, about 6.7 to about 7.5, about 6.7 to about 7.3, about 6.7 to about 7.1, about 6.9 to about 8, about 6.9 to about 7.7, about 6.9 to about 7.5, about 6.9 to about 7.3, about 6.9 to about 7.1, or about 7.

[0178] Preferably, the method further comprises diluting the complex using a buffer having a pH of from 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9 to 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, preferably about 7. Preferably, the method further comprises diluting the complex with a buffer having a pH of 6 to 8, 6 to 7.7, 6 to 7.5, 6 to 7.3, 6 to 7.1, 6.3 to 8, 6.3 to 7.7, 6.3 to 7.5, 6.3 to 7.3, 6.3 to 7.1, 6.5 to 8, 6.5 to 7.7, 6.5 to 7.5, 6.5 to 7.3, 6.5 to 7.1, 6.7 to 8, 6.7 to 7.7, 6.7 to 7.5, 6.7 to 7.3, 6.7 to 7.1, 6.9 to 8, 6.9 to 7.7, 6.9 to 7.5, 6.9 to 7.3, 6.9 to 7.1, or about 7.

[0179] Preferably, the buffer comprises bovine serum albumin, preferably 0.1% to 1.0% weight / volume bovine serum albumin. Preferably, the buffer comprises a non-ionic detergent, preferably a non-ionic detergent that does not substantially denature or aggregate the protein. Preferably, the buffer comprises a non-denaturing non-ionic detergent. Various non-denaturing non-ionic detergents are known in the art and may be suitably selected, examples being Tween-20, Tween-80, digitonin, n-dodecyl-β-D-maltoside, Triton X-100, Triton X-114, Nonidet P-40 (NP-40), CA-630. Optionally, the buffer preferably contains a non-denaturing ionic detergent such as bile acid.

[0180] The present invention also relates to an ex vivo method of diagnosing a disease or condition in a subject, comprising determining the presence or absence of a target DNA sequence associated with a disease or condition according to the present invention in a sample obtained from the subject, wherein the presence indicates a diagnosis of the disease or condition in the subject.

[0181] The present invention relates to an ex vivo method for diagnosing a disease or condition in a subject, comprising:

[0182] a. determining the presence or absence of a target DNA sequence associated with a disease or disorder according to the present invention in a sample obtained from a subject;

[0183] b. comparing the presence or absence determined in step a with the presence or absence of a target DNA sequence associated with a disease or disorder in a sample from an individual not suffering from the disease or disorder, in a negative control, or in a positive control determined according to the present invention;

[0184] c. detecting the presence of differences in the target DNA sequences of the subjects compared in step b; and

[0185] d. determining the extent of the difference, wherein if samples from individuals not suffering from the disease or condition or negative controls are compared, a difference greater than a predetermined threshold indicates a diagnosis of the disease or condition in the subject, wherein if a positive control is compared, no difference, a difference greater than a predetermined threshold, or a difference less than a predetermined threshold indicates a diagnosis of the disease or condition in the subject.

[0186] The predetermined threshold may be suitably set by the skilled person and may, for example, depend on the background signal of the particular amplification and / or detection method used or on the amount of target DNA sequence relevant to the diagnosis of the disease or disorder.The subject or individual may be a human or an animal.

[0187] According to the present invention, preferably, the control is a control sample. The control sample may or may not contain a target DNA sequence associated with a disease or condition, thereby being a positive control sample or a negative control sample, respectively. Preferably, the control sample is a sample not obtained from a subject or individual. Preferably, the control comprises reference data. Preferably, the reference data comprises data obtained from a sample comprising a target DNA sequence associated with a disease or condition, preferably wherein the sample is obtained from a subject suffering from a disease or condition.

[0188] According to the present invention, preferably, the disease or condition comprises cancer, an autoimmune disease or condition, a genetic disease or condition, pain, a respiratory disease or condition, a cardiovascular disease or condition, an infectious disease or condition, a thyroid disease or condition, a skin disease or condition, diabetes, obesity, a kidney disease or condition, a gastrointestinal disease or condition, a neurodegenerative disease or condition, a musculoskeletal disease or condition, a metabolic disease or condition, or a psychiatric disorder, preferably cancer, an autoimmune disease or condition, or an infectious disease or condition.

[0189] Preferably, the target DNA sequence associated with a disease or condition or its onset comprises a DNA sequence expressing a tumor antigen, a tumor marker or a tumor suppressor. Preferably, the target DNA sequence associated with a disease or condition or its onset comprises a DNA sequence having a nucleotide sequence polymorphism. Preferably, the nucleotide sequence polymorphism comprises one or more somatic mutations and / or single nucleotide polymorphisms. Preferably, the target DNA sequence associated with a disease or condition or its onset comprises a DNA sequence associated with an epigenetic event, preferably DNA methylation and / or DNA hydroxymethylation.

[0190] According to the present invention, preferably, disease or illness include infectious diseases or illness, more preferably sexually transmitted infection. Preferably, disease or illness include chlamydia, gonorrhea, syphilis, genital herpes, acquired immunodeficiency syndrome, papilloma or trichomoniasis. Preferably, the target DNA sequence relevant to disease or illness includes bacterium Chlamydia trachomatis (Chlamydiatrachomatis), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Treponema pallidum (Treponemapallidum), virus human alpha herpesvirus 1, human alpha herpesvirus 2, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus or the sequence of parasite Trichomonas vaginalis (Trichomonas vaginalis).

[0191] The subject can be human or animal. The sample obtained from the subject can include tissue and / or biological fluid. Such a sample can be obtained in vitro, in vitro or in vivo. As a non-limiting example, the sample obtained from the subject can be selected from the tissue, organ, cell or any separated fraction of a human or animal subject. The sample obtained from the subject can also be selected from blood, plasma, lymph, saliva, urine, feces, tears, sweat, sperm, vaginal fluid or cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid or pericardial fluid, and any fraction or extract thereof. The sample can be obtained by any technology known in the art.

[0192] The sample obtained from the subject can be subjected to a treatment step before step a of the diagnostic method according to the present invention. The treatment can help determine the presence or absence of a target DNA sequence associated with a disease or condition. Various treatment methods are known in the art. For example, heat, mechanical or chemical treatment of the sample obtained from the subject can lyse the cells, thereby releasing DNA from the cells. Some nucleic acid amplification methods, such as RPA, can then be performed directly in certain biological samples (milk, urine, feces, pleural fluid) after heat, mechanical or chemical lysis.

[0193] The present invention also relates to a method for detecting a nucleic acid amplification product comprising a target DNA sequence, the method comprising:

[0194] a. contacting the nucleic acid amplification product with a recombinase and an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a label, thereby forming a D-loop comprising triple-stranded DNA, the probe and the recombinase in the nucleic acid amplification product; and

[0195] b. Detecting nucleic acid amplification products, including detection probes.

[0196] As discussed above, the inventors surprisingly found that they could detect a nucleic acid amplification product comprising a target DNA sequence and a D-loop, the D-loop comprising a recombinase and an oligonucleotide probe containing a nucleic acid sequence complementary to the target DNA sequence and a label. The triple-stranded DNA in the D-loop is formed by the recombinase and the probe, which can then be used to detect the nucleic acid amplification product via the label of the detection probe.

[0197] It should be understood that, where possible, any preferred features or steps of the methods disclosed in the present application are also preferably applicable to the above-mentioned method for detecting a nucleic acid amplification product comprising a target DNA sequence. For example, the probe may preferably comprise a first label or a second label according to the present invention, the probe may preferably comprise a polymerase blocking group according to the present invention, the recombinase may preferably be a recombinase selected from the group consisting of: RecA, DrRecA, Rad51, RadA or UvsX recombinase or a homologue thereof, the method may preferably further comprise adding an ATP analogue, more preferably a non-hydrolyzable or hydrolyzable ATP analogue, etc.

[0198] Preferably, according to the present invention, detecting the nucleic acid amplification product comprises combining a label of a probe that binds to the nucleic acid amplification product with a binding agent. Preferably, the binding agent is attached to a solid support. Preferably, detecting the nucleic acid amplification product comprises using a lateral flow test, a lateral flow device, an enzyme-linked immunosorbent assay, a magnetic immunoassay or a flocculation assay.

[0199] According to any of the methods of the invention, preferably the method is nuclease-free or protease-free. Preferably, the method is nuclease-free and protease-free.

[0200] The present invention provides devices and systems for amplifying target DNA sequences and / or detecting nucleic acid amplification products of the present invention. Devices and systems may include other features that enable or facilitate their intended use in combination with the disclosed features of the method of the present invention. Devices and systems according to the present invention are suitable for methods according to the present invention.

[0201] The present invention relates to a nucleic acid amplification system, comprising:

[0202] A first primer and a second primer for amplifying a target DNA sequence, a DNA-dependent DNA polymerase, and a dNTP monomer containing a first label. Preferably, the system also comprises a recombinase. Preferably, the system also comprises an oligonucleotide probe, which comprises a nucleic acid sequence complementary to the target DNA sequence and a second label.

[0203] The invention relates to a nucleic acid detection system for detecting a target DNA sequence, comprising: a recombinase, an oligonucleotide probe containing a nucleic acid sequence complementary to the target DNA sequence and a second marker, and a nucleic acid amplification product containing the target DNA sequence and nucleotides containing the first marker.

[0204] The present invention relates to a nucleic acid amplification system, comprising:

[0205] A first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and hybridizes with the first strand of DNA of the target DNA sequence, and a DNA-dependent DNA polymerase. Preferably, the system further comprises a recombinase. Preferably, the system further comprises an oligonucleotide probe, the oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first label, preferably wherein the probe hybridizes with the first strand or the second strand of DNA of the target DNA sequence, and the second strand is complementary to the first strand.

[0206] The present invention relates to a nucleic acid detection system for detecting a target DNA sequence, which comprises: a recombinase, an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label, a nucleic acid amplification product comprising the target DNA sequence, and a primer containing a second label and hybridizing with a DNA chain of the target DNA sequence.

[0207] The present invention relates to a lateral flow device comprising a lateral flow strip, wherein the lateral flow strip comprises:

[0208] - sample application zone;

[0209] - a reagent zone downstream of and in fluid communication with the sample application zone, wherein the reagent zone comprises a non-immobilized first binding agent bound to a first label;

[0210] - a test zone downstream of and in fluid communication with the reagent zone, wherein the test zone comprises an immobilized second binding agent bound to a second label; and

[0211] - a control zone downstream of the test zone and in fluid communication with the test zone.

[0212] Preferably, the reagent zone comprises a dried, preferably lyophilized, composition for amplifying a target DNA sequence. Preferably, the composition is a composition for RPA. Preferably, the composition is the first composition or the second composition according to the present invention.

[0213] The present invention also relates to a first component kit comprising a dNTP monomer and an oligonucleotide probe, wherein the dNTP monomer contains a first label, and the oligonucleotide probe comprises a nucleic acid sequence complementary to a target DNA sequence and a second label. Preferably, the component kit is used for amplification of nucleic acids, preferably wherein the amplification is a recombinase polymerase amplification (RPA). Preferably, the component kit comprises a recombinase. Preferably, the component kit comprises a DNA-dependent polymerase. Preferably, the component kit comprises a first primer and a second primer for amplifying a target DNA sequence. Preferably, the component kit comprises a lateral flow device or a device according to the present invention. The first component kit according to the present invention may also comprise the features of the first method of the present invention or the features of the second method of the present invention as much as possible.

[0214] The present invention also relates to a second component kit, which comprises a first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and hybridizes with the first strand of the DNA of the target DNA sequence; and an oligonucleotide probe, which comprises a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing the first label, preferably, wherein the probe hybridizes with the first strand or the second strand of the DNA of the target DNA sequence, and the second strand is complementary to the first strand. Preferably, the component kit is used for amplification of nucleic acids, preferably wherein the amplification is a recombinase polymerase amplification (RPA). Preferably, the component kit comprises a recombinase. Preferably, the component kit comprises a DNA-dependent polymerase. Preferably, the component kit comprises a lateral flow device or a device according to the present invention. The second component kit according to the present invention may also comprise the features of the second method of the present invention or the features of the first method of the present invention as much as possible.

[0215] Preferably, the kit of parts according to the invention is nuclease-free or protease-free. Preferably, the kit of parts is nuclease-free and protease-free. Suitably, the kit of parts is ATP-free or ATP analog-free, preferably comprising DrRecA. Optionally, the kit of parts according to the invention comprises instructions for carrying out the method according to the invention. Preferably, the kit of parts according to the invention is for use in the method according to the invention.

[0216] In addition, the present invention relates to the use of a device, system or component kit according to the present invention for determining the presence or absence of a pathogen or target DNA sequence associated with a disease or condition or its onset. Preferably, the pathogen is a human, animal or plant pathogen. Preferably, the disease or condition or its onset includes cancer, autoimmune disease or condition, genetic disease or condition, pain, respiratory disease or condition, cardiovascular disease or condition, infectious disease or condition, thyroid disease or condition, skin disease or condition, diabetes, obesity, kidney disease or condition, gastrointestinal disease or condition, neurodegenerative disease or condition, musculoskeletal disease or condition, metabolic disease or condition, or mental illness, more preferably cancer, autoimmune disease or condition, or infectious disease or condition. Preferably, the target DNA sequence associated with a disease or condition or its onset includes a DNA sequence expressing a tumor antigen, a tumor marker or a tumor suppressor. Preferably, the target DNA sequence associated with a disease or condition or its onset includes a DNA sequence having a nucleotide sequence polymorphism. Preferably, the nucleotide sequence polymorphism includes one or more somatic mutations and / or single nucleotide polymorphisms. Preferably, the target DNA sequence associated with a disease or disorder or the onset thereof comprises a DNA sequence associated with an epigenetic event, preferably DNA methylation and / or DNA hydroxymethylation.

[0217] The device, system or component kit according to the present invention can be used to determine the presence or absence of a target DNA sequence in various types of samples. For example, these include but are not limited to diagnostic samples, veterinary samples, pharmaceutical samples, cosmetic samples, food samples, beverage samples, feed samples, agricultural samples, soil samples, air samples, water samples, waste samples, effluent samples or sewage samples.

[0218] The present invention relates to the use of a device, a system or a component kit according to the present invention for determining the presence or absence of a target DNA sequence in a diagnostic sample, a veterinary sample, a pharmaceutical sample, a cosmetic sample, a food sample, a beverage sample, a feed sample, an agricultural sample, a soil sample, an air sample, a water sample, a waste sample, an effluent sample or a sewage sample.

[0219] Detailed description of the drawings

[0220] The present invention will now be discussed with reference to the accompanying drawings, which show preferred exemplary embodiments of the invention.

[0221] Figure 1A schematic depiction of a first method of the present invention is shown. A sample comprising a DNA molecule (100) for determining the presence or absence of a target DNA sequence is provided, wherein the target DNA sequence is included. The sample is contacted with: a first primer (101) and a second primer (102) for amplifying the target DNA sequence; a recombinase (not shown); a DNA-dependent DNA polymerase (not shown); a dNTP containing a first label (103); and an oligonucleotide probe (104) comprising a nucleic acid sequence complementary to the target DNA sequence and a second label (105). Optionally, the probe comprises a polymerase blocking group (106), preferably located at the 3' end of the probe, such as Figure 1 . The blocking group (106) prevents the polymerase extension of the primer. The target DNA sequence is amplified (107) by, for example, PCR or RPA to produce a nucleic acid amplification product (110), wherein the dNTP (103) containing the first label has been incorporated into the nucleic acid amplification product (110). The probe (104) and the recombinase can also be added to the sample after amplification (107), which can be their only addition or addition in addition to the first addition. The recombinase promotes the formation of a DNA triplex (111) between the target DNA sequence and the probe. The nucleic acid amplification product is then detected via, for example, flocculation (108), lateral flow strips (109) or ELISA (not shown).

[0222] In short, detection via flocculation (108) comprises combining a nucleic acid amplification product (110) and a first binding agent (112) capable of binding a first label, wherein the product and the first binding agent are capable of forming a visually detectable complex, wherein the complex is formed via flocculation, preferably by flocculation of the first binding agent. The first binding agent preferably specifically binds to the first label. Here, if the first label is biotin, the first binding agent (112) can be a gold nanoparticle coated with streptavidin, because the large amount of the first label incorporated, the flocculation of the gold nanoparticles enables visual detection of the nucleic acid amplification product.

[0223] In short, detection via a lateral flow strip (109) includes providing a lateral flow strip comprising a first binding agent (112) capable of binding a first label and a second binding agent (115) capable of binding a second label, preferably wherein the second binding agent is fixed to the lateral flow strip. The lateral flow strip typically comprises a nitrocellulose membrane (116) and an end zone (118). The nucleic acid amplification product (110) is applied to the sample application zone (113) of the lateral flow strip. The product moves along the strip, passing through a reagent zone (114) comprising a first binding agent (112) capable of binding a first label, where the first binding agent binds to the first label incorporated into the nucleic acid amplification product. The product moves further along the strip to a test zone, where it binds to a fixed second binding agent (115), which is capable of binding to the second label if the probe (104) has been bound to the product. This then enables detection of the nucleic acid amplification product. A control zone comprising a binding agent (117) capable of binding to the first binding agent binds to the remaining first binding agent. Here, if the first label is biotin, the first binding agent (112) can be gold nanoparticles coated with streptavidin. The combination of the gold nanoparticles coated with streptavidin and the first label incorporated in large quantities enables visual detection with high sensitivity. The probe (114) that binds to the product enables high specificity for the detected product.

[0224] Figure 2 A schematic depiction of a second method of the present invention is shown. A sample comprising a DNA molecule (100) for determining the presence or absence of a target DNA sequence is provided, wherein the target DNA sequence is included. The sample is contacted with: a first primer (101) and a second primer (102) for amplifying the target DNA sequence, wherein the second primer contains a second label (105); a recombinase (not shown); a DNA-dependent DNA polymerase (not shown); dNTPs; and an oligonucleotide probe (104) comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label (103). Optionally, the probe comprises a polymerase blocking group (106), preferably located at the 3' end of the probe, such as Figure 2 . The blocking group (106) prevents the polymerase extension of the primer. The target DNA sequence is amplified (107) by, for example, PCR or RPA to produce a nucleic acid amplification product (110). The probe (104) and recombinase can also be added to the sample after amplification (107), which can be their only addition or addition in addition to the first addition. The recombinase promotes the formation of a DNA triplex (111) between the target DNA sequence and the probe. The nucleic acid amplification product is then detected via, for example, flocculation (108), lateral flow strips (109) or ELISA (not shown).

[0225] In short, detection via flocculation (108) includes combining a nucleic acid amplification product (110) and a first binding agent (112) capable of binding to a first label, wherein the product and the first binding agent are capable of forming a visually detectable complex, wherein the complex is formed via flocculation, preferably by flocculation of the first binding agent. The first binding agent preferably specifically binds to the first label. Here, if the first label is biotin, the first binding agent (112) can be streptavidin-coated, because a large amount of the first label is bound to the product via the probe, and flocculation of the gold nanoparticles enables visual detection of the nucleic acid amplification product.

[0226] In short, detection via a lateral flow strip (109) includes providing a lateral flow strip comprising a first binding agent (112) capable of binding a first label and a second binding agent (115) capable of binding a second label, preferably wherein the second binding agent is fixed on the lateral flow strip. The lateral flow strip typically comprises a nitrocellulose membrane (116) and an end zone (118). The nucleic acid amplification product (110) is applied to the sample application zone (113) of the lateral flow strip. The product moves along the strip, passing through a reagent zone (114) comprising a first binding agent (112) capable of binding a first label, and if the probe has been bound to the product, the first binding agent is bound to the first label incorporated into the probe (104) on the reagent zone. The product moves further along the strip to a test zone, where it is bound to a fixed second binding agent (115) capable of binding a second primer (102). This then enables detection of the nucleic acid amplification product. A control zone comprising a binding agent (117) capable of binding a first binding agent is bound to the remaining first binding agent. Here, if the first label is biotin, the first binding agent (112) can be gold nanoparticles coated with streptavidin. The combination of the gold nanoparticles coated with streptavidin and the first label incorporated in large quantities enables visual detection with high sensitivity. The probe (114) that binds to the product enables high specificity for the detected product.

[0227] Figure 3 The effect of biotin-dCTP concentration on RPA yield is shown. A real-time assay was used to assess the effect of biotin-16-dCTP incorporation during recombinase polymerase amplification. Figure 3 A shows amplification with different biotin-16-dCTP concentrations (mol %) of the total dCTP used, i.e. 0%, 20%, 50% and 70% molar biotin-16-dCTP of the total dCTP. The fluorescence indicating double-stranded DNA formation was measured every 30 seconds. The y-axis indicates relative fluorescence and the x-axis indicates time in minutes. Further details of the experiment are described in Example 1. The maximum tolerable amount was found to be about 50% molar biotin-16-dCTP of the total dCTP. Figure 3B shows biotin-16-dCTP compared to Figure 3 % dose response of the area under the curve (AUC) of the amplification curve of A. The y-axis indicates AUC and the x-axis indicates the different percentages of biotin-16-dCTP to total dCTP in % mol. The half maximal response (IC50) was found to be 52.5%.

[0228] Figure 4 The effect of biotin-dUTP concentration on RPA yield is shown. A real-time assay was used to assess the effect of biotin-16-dUTP incorporation during recombinase polymerase amplification. Figure 4 A shows amplification with different biotin-16-dUTP concentrations (mol %) of the total dUTP used, i.e., 0%, 20%, 50% and 70% molar biotin-16-dUTP of the total dUTP. The fluorescence indicating double-stranded DNA formation was measured every 30 seconds. The y-axis indicates relative fluorescence and the x-axis indicates time in minutes. Further details of the experiment are described in Example 2. It was found that the maximum tolerable amount was about 50% molar biotin-16-dUTP of the total dUTP. Figure 4 B shows biotin-16-dUTP compared to Figure 4 % dose response of the area under the curve (AUC) of the amplification curve of A. The y-axis indicates AUC and the x-axis indicates the different percentages of biotin-16-dUTP to total dUTP in % mol. The half maximal response (IC50) was found to be 55.5%.

[0229] Figure 5 The effect of both biotin-dCTP and biotin-dUTP concentrations on RPA yield is shown. A real-time assay was used to assess the effect of biotin-16-dCTP and biotin-16-dUTP incorporation during recombinase polymerase amplification to maximize the amount of biotin per amplicon. Figure 5 Real-time RPA amplification of six different conditions is shown, wherein 0%, 50% C and 0% U, 0% U and 50% C, 40% C and 60% U, 50% C and 50% U, and 60% C and 40% U are used, n=2. "C" indicates the mol / mol of biotin-16-dCTP in the total dCTP in a single reaction, and "U" indicates the mol / mol of biotin-16-dUTP in the total dUTP in a single reaction. The fluorescence indicating the formation of double-stranded DNA is measured every 30 seconds. The y-axis indicates relative fluorescence, and the x-axis indicates time in minutes. Further details of the experiment are described in Example 3. When a combination of 50% biotin-16-dUTP and 40% biotin-16-dCTP and 60% biotin-16-dUTP is used, the maximum yield is observed.

[0230] Figure 6Evidence for D-loop formation on an agarose gel is shown. Figure 6 The results of reactions containing different components are shown: lane 1 contains only homologous DNA, lane 2 contains only primers, lane 3 contains homologous DNA, primers and RecA, lane 4 contains homologous DNA, primers, RecA and ATPγS, and lane 5 contains a 100bp DNA ladder, wherein the bottom band indicates 100bp, and the band above the bottom band indicates 200bp. Homologous DNA contains a sequence complementary to the primer, which is used as a probe according to the present invention. Further details of the experiment are described in Example 5. Non-denaturing agarose gel shows the formation of a stable D ring (lane 4) in the presence of ATPγS, primers, RecA and homologous DNA. It is known that ATPγS stabilizes the D ring, which is observed as a tail in lane 4.

[0231] Figure 7 Evidence for D-loop formation in ELISA is shown. A high throughput D-loop ELISA assay was developed that mimics a lateral flow setup, the details of which are described in Example 6. Figure 7 A illustrates the concept of ELISA. Here, an antibody (115), i.e., a second binding agent according to the present invention, is immobilized, which binds to the 5'-label (105) of the probe, which is a second label according to the present invention. A nucleic acid amplification product (110) containing nucleotides with a first label (103) produced according to the first method of the present invention is contacted with a recombinase and a probe. Subsequently, a D loop (111) is formed, and the complex containing the nucleic acid amplification product (110) and the probe is incubated with the immobilized antibody (115). After several conventional washing steps, the bound complex is contacted with a streptavidin-horseradish peroxidase (HRP) fusion (120) and then detected.

[0232] Figure 7 B shows the use of Figure 7 Experiment of the concept illustrated in A. The left column shows the absorbance using homologous DNA containing 50% mol biotin-16-dUTP, while the right column shows the absorbance when the homologous DNA is replaced by HO. The y-axis indicates the absorbance at a wavelength of 450 nm. The homologous DNA contains a sequence complementary to the primer, which is used here as a probe according to the present invention. A two-fold increase in the signal is observed in the presence of the homologous biotinylated DNA.

[0233] Figure 8 Evidence for D-loop formation in a lateral flow assay is shown. The possibility of using stable D-loop formation to detect DNA on lateral flow strips was evaluated. Figure 8 The concept of detection is illustrated in Figure A. Figure 1, where the same symbols are used. The nucleic acid amplification product containing the first label and the incorporated nucleotide is contacted with a probe containing the second label (i.e., FAM) and a recombinase. The resulting complex containing the D ring is then contacted with a lateral flow strip and detected at the FAM binding site.

[0234] Figure 8 B shows a comparison of the use of ATPγS compared to H2O during contact of a nucleic acid amplification product with a probe containing a second label FAM and a recombinase. It is known that ATPγS stabilizes the D-loop in combination with the recombinase. "D" indicates a digoxigenin binding site, "F" indicates a FAM binding site, and "S" indicates a streptavidin binding site. The top bar shows the results of an experiment using ATPγS, while the bottom bar shows the results of an experiment in which ATPγS was replaced with H2O. Further details of the experiment are described in Example 7. The band on the FAM binding site of the top bar indicates that ATPγS increases the detection of a complex containing a nucleic acid amplification product and a probe.

[0235] Fig. 9 The effect of probe design on lateral flow assays is shown. The stability of D-loop formation in various probe designs was evaluated as a function of their design, which refers to binding to the end of the biotinylated homologous DNA ( Fig. 9 A and Fig. 9 B) or middle ( Fig. 9 C. Fig. 9 D. Fig. 9 E and Fig. 9 F) probe, wherein Fig. 9 C and Fig. 9 A 25-nucleotide (nt) probe was evaluated in D and Fig. 9 E and Fig. 9 A 60 nt probe was evaluated in F. The homologous DNA contained a sequence complementary to the probe. In addition, several conditions known to stabilize D-loops were tried because D-loops formed at the ends (i.e., plectonemic joints) are more stable than D-loops formed in the middle of a DNA duplex (i.e., parallel sliding joints). These conditions were the presence of single-stranded binding proteins, PEG, KCl, trehalose, the ionic liquid 1-ethyl-3-methylimidazolium acetate, and combinations thereof. The results showed that the end probes were able to bind regardless of the conditions, while the binding of the middle binding probes varied with their length and additives. The best results were achieved with the 60 nt probe in the presence of ionic liquids, see Fig. 9 F. Further details of the experiment are described in Example 8.

[0236] Fig. 9 A. Fig. 9 C and Fig. 9 E shows a schematic diagram of the detection concept of different probes, ref. Figure 1, where the same symbols are used. A nucleic acid amplification product containing a first labeled incorporated nucleotide is contacted with a probe containing a second label (i.e., digoxigenin) and a recombinase and optionally other additives. The resulting complex containing the D-loop is then contacted with a lateral flow strip and detected at the digoxigenin binding site.

[0237] Fig. 9 B. Fig. 9 D and Fig. 9 F shows the lateral flow assay Fig. 9 A. Fig. 9 C and Fig. 9 E. "D" indicates digoxigenin binding site, "F" indicates FAM binding site, and "S" indicates streptavidin binding site. Bar 1 indicates no additives, bar 2 indicates added single-chain binding protein, bar 3 indicates added trehalose, PEG 6000 and KCl, bar 4 indicates added ionic liquid 1-ethyl-3-methylimidazolium acetate, and bar 5 indicates added single-chain binding protein, trehalose, PEG, KCl and ionic liquid 1-ethyl-3-methylimidazolium acetate. Further details of the experiment are described in Example 8. Fig. 9 B and Fig. 9 The bars of F, the terminal probe and the middle 60 nt probe, respectively, show a band in each digoxigenin binding site, indicating that a complex containing the nucleic acid amplification product and the probe was detected.

[0238] Fig.10 The specificity of primer-probes and probes with and without ATP-γ-S is shown and also compared with a conventional hapten-based strategy. The extent of sequence-specific binding of the 29nt primer probe to the ends of the amplicons of templates amplified and purified by RPA using 50% biotin-16-dUTP and the 60nt probe to the middle of the amplicon of templates amplified and purified by RPA using 50% biotin-16-dUTP was evaluated. Comparisons were also made with RPA amplifications with conventional hapten-labeled primers. The effects of replacing ATP-γ-S with limited amounts of ATP and ATP with an ATP regeneration system (phosphocreatine and creatine kinase) and its combination with ATP-γ-S were plotted.

[0239] Fig.10A shows the specificity of primer-probe (helical winding connection) D-loop detection using ELISA. It shows the results of experiments in which a homologous DNA template was amplified using appropriate primers, generating a reference "Ho", or in which a different non-homologous DNA template was amplified using appropriate primers, generating a reference "No". The amplified DNA was subjected to primer-probe (indicated by "Pri") and recombinase incubation using different ATP / ATPγS inputs. Here, homologous and non-homologous refer to sequences complementary to the primer-probe. The different ATP / ATPγS inputs are: ATP regeneration via ATP with creatine kinase and phosphocreatine (1x basic E-mix), indicated by "rA"; ATPγS alone, indicated by "Aγ"; ATP alone, indicated by "A"; a combination of rA and Aγ, indicated by "rA / Aγ"; a combination of A and Aγ, indicated by "A / Aγ". The resulting D-loop was then detected using ELISA. Further details of the experiment are described in Example 9.

[0240] Since the primer-probe is not complementary to the non-homologous DNA template No, a D-loop should not form, resulting in a signal that can be considered a negative signal. In contrast, the primer-probe is complementary to the DNA template Ho, resulting in a D-loop and a positive signal. Fig.10 It can be observed in A that for each ATP / ATPγS input condition, the positive signal is higher than the negative signal, confirming the presence of the D-loop. The greater difference between the negative and positive signals for the corresponding ATP / ATPγS input condition can be considered as a greater specificity of the probe for the corresponding condition. Fig.10 In A, the greatest specificity was observed for the (Pri-)rA / Aγ condition, while the lowest specificity was observed for the (Pri-)A condition. In general, higher specificity was observed when ATPγS was used.

[0241] Fig.10B shows the specificity of probe-based (parallel sliding connection) D-loop detection using ELISA. It shows the results of experiments in which a homologous DNA template was amplified using appropriate primers to generate a reference "Ho", or in which a different non-homologous DNA template was amplified using appropriate primers to generate a reference "No". The amplified DNA was subjected to a 60nt probe (indicated by "Pro") and a recombinase incubation using different ATP / ATPγS inputs. Here, homologous and non-homologous refer to sequences complementary to the primer-probe. Different ATP / ATPγS inputs are: ATP regeneration via ATP with creatine kinase and phosphocreatine (1x basic E-mix), indicated by "rA"; ATPγS alone, indicated by "Aγ"; ATP alone, indicated by "A"; a combination of rA and Aγ, indicated by "rA / Aγ"; a combination of A and Aγ, indicated by "A / Aγ". The resulting D-loop was then detected using ELISA. Further details of the experiment are described in Example 9.

[0242] Since the probe is not complementary to the non-homologous DNA template No, a D-loop should not form, resulting in a signal that can be considered a negative signal. In contrast, the probe is complementary to the DNA template Ho, resulting in a D-loop and a positive signal. Fig.10 It can be observed in B that for each ATP / ATPγS input condition, the positive signal is higher than the negative signal, confirming the presence of the D-loop. The greater difference between the negative and positive signals for the corresponding ATP / ATPγS input condition can be considered as a greater specificity of the probe for the corresponding condition. Fig.10 In B, the greatest specificity is observed for the (Pro-)A / Aγ condition, while the lowest specificity is observed for the (Pro-)rA condition. In general, higher specificity is observed when ATPγS is used.

[0243] In addition, comparison Fig.10 A and Fig.10 B, it can be observed that the probe-based D-loop detection has greater specificity than the primer-probe-based D-loop detection. This is surprising because D-loops formed at the ends (i.e., helical winding junctions) are more stable than D-loops formed in the middle of a DNA duplex (i.e., parallel sliding junctions).

[0244] Fig.10 C shows a comparison of the specificity of D-loop detection using ELISA with conventional double hapten labeled RPA amplicons. Conditions Pro-No-A / Aγ, Pro-Ho-A / Aγ, Pri-No-A / Aγ, and Pri-Ho-A / Aγ were taken from Fig.10 B and Fig.10Experiment described in A. In addition, two conventional double hapten-labeled RPA amplicons were generated as follows: the homologous and non-homologous DNA templates were amplified using a biotin-labeled forward primer and a digoxigenin-labeled reverse primer, respectively, to generate references "5'DIG-5'bio-Ho" (specific amplification product of RPA) and "5'DIG-5'bio-No" (i.e., non-specific amplification product of RPA), respectively. Fig.10 A and Fig.10 The products were detected by ELISA under the same conditions as described in B. Further details of the experiments are described in Example 9.

[0245] It can be observed that D-loop detection according to the present invention, especially when a (stable) parallel sliding junction is formed, produces an improved signal compared to detection of RPA-amplified DNA by conventional double hapten labeling, where detection of RPA-amplified DNA by conventional double hapten labeling produces a lower relative signal (i.e., the signal of 5'DIG-5'bio-Ho minus the signal of 5'DIG-5'bio-No).

[0246] Fig.11 It is shown that the RPA amplicon can be directly added to the immobilized D-ring detection in conjunction with the RecA probe, which has high repeatability and specificity. The serial dilutions of the crude RPA reaction in deionized water are added to the RecA reaction in triplicate with 0.3mM ATPγS and probe_2 in 1 microliter volume, and then added to ELISA in triplicate (Pro-HO-Aγ, crude, [dilution factor 1x, 2x or 4x]). Negative controls (Pro-NO-Aγ) and positive controls (5'DIG-5'bio-Ho and Pro-HO-Aγ) are also used. As a specificity test, Non_1 is amplified in RPA with 30% biotin-16-dUTP, and then the crude RPA reaction is serially diluted in deionized water. The serial dilutions were added to triplicate RecA reactions with 0.3 mM ATPγS and probe_2 in 1 microliter volume and subsequently added to ELISA in triplicate (Pro-NO-Aγ, crude, [dilution factor 1x, 2x or 4x]). Comparison between serial dilutions of Hom_1 and Non_1 amplicons in crude RPA reactions shows that the specificity and repeatability of the D-loop-based detection method are retained even after the crude RPA product is added directly to the RecA reaction. Further details of the experiment are described in Example 10.

[0247] Fig.12 This showed that DrRecA does not require ATP or ATPγS for strand exchange. Real-time fluorescence assay for strand exchange activity of the recombinase ( Fig.12A). Strand exchange activity of the RecA enzyme from Deinococcus radiodurans (DrRecA) was detected and was independent of the presence of an ATP source. This was confirmed by comparing the fluorescence intensity of the real-time fluorescence assay products on an agarose gel ( Fig.12 B). The presence of fluorescence upon addition of DrRecA without an ATP source indicates strand exchange without the need for ATP and its cofactors, which can simplify the reaction composition of the RecA reaction. Further details of the experiment are described in Example 11.

[0248] Fig.13 It is shown that bridge flocculation allows for the naked eye detection of biotinylated DNA. Bridge flocculation was performed on a sample comprising a purified RPA product comprising a biotinylated DNA amplicon according to the present invention. Significant differences in color development between samples treated with purified RPA product with and without template DNA can be observed, indicating that this method can be used to detect biotinylated RPA amplicons with the naked eye after RPA. Further details of the experiment are described in Example 12.

[0249] Fig.14 Stable D-loop formation of EcRecA and DrRecA is shown. Stable D-loop formation was analyzed using ELISA using different ATP compositions: No ATP ATP (A), dATP (dA), ATPγS (Aγ) and ATP regeneration system (rA). Further details of the experiment are described in Example 13.

[0250] Throughout the description and claims of this specification, the words "comprises / includes" and "comprising" and variations thereof mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0251] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating more than one as well as the singular, unless the context otherwise requires.

[0252] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible with that aspect, embodiment or example.

[0253] The patents, scientific and technical literature referred to herein establish the knowledge available to those skilled in the art as of the filing date. The entire contents of issued patents, published and pending patent applications, and other publications cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the event of any inconsistency, the present disclosure will control.

[0254] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2nd Edition, John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary of many terms used in the present invention for those skilled in the art. Although any methods and materials similar to or equivalent to those described herein can be used in practicing the present invention, preferred methods and materials are described herein. Therefore, the terms to be defined below are more fully described by reference to the specification as a whole. In addition, unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" as used herein include plural indicators. Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described as these may vary depending upon the circumstances in which they are used by one skilled in the art.

[0255] All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0256] Unless expressly stated otherwise, each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0257] The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination of steps in any method or process so disclosed.

[0258] Aspects of the invention are illustrated by the following non-limiting examples. Example

[0259] General Materials and Methods

[0260] Unless otherwise stated, the following materials and methods were used in the examples.

[0261] The method of the embodiments is nuclease-free and protease-free: no nucleases or proteases are used.

[0262] Material

[0263] Primers (see Table 1), probes (see Table 2), and synthetic target and non-target double-stranded DNA templates (see Table 3) were purchased from IDT and Biolegio. TwistDx liquid kit was purchased from TwistDx. PCR reaction components, RecA, RecA reaction buffer, T4 gene 32 protein, Gel Loading Dye, Purple (6X, without sodium dodecyl sulfate (SDS)), and 100 bp DNA ladder were obtained from New England Biolabs. ATPγ-S was purchased from Abcam. SYBR Safe, Qubit assays, and E-Gel PowerSnap electrophoresis systems were purchased from ThermoFisher Scientific. Tissue culture (TC) treated flat bottom clear 96-well ELISA plates were obtained from Greiner. PCRD FLEX lateral flow strips and PCRD FLEX extraction buffer were provided by Abington Health. Biotin-16-dCTP was purchased from Jena Biosciences. Anti-digoxigenin antibody and anti-fluorescein antibody were purchased from Enzo LifeSciences. Unless otherwise stated, agarose, salts, 3,3',5,5'-tetramethylbenzidine (TMB), dithiothreitol (DTT), H2O2, H2SO4, bovine serum albumin (BSA), and buffers were purchased from Fisher Scientific and Merck.

[0264] Table 1. Primers

[0265]

[0266] Table 2. Probes

[0267]

[0268] Table 3. Synthetic DNA templates

[0269]

[0270]

[0271] Primer design and validation

[0272] Target and non-target DNA template sequences were selected according to the requirements outlined by TwistDx: GC content between 40% and 60%, few repetitive sequences, and absence of forward / reverse repeats and palindromes. Primer pairs for target sequences were designed using PrimedRPA (Higgins et al., accessed on February 7, 2022 via GitHub (Higgins, M. PrimedRPA, 2022)).

[0273] The resulting candidate primer pairs were validated by RPA using the TwistDx Liquid Basic kit according to the manufacturer's protocol, purified with Zymogen DNA Clean & Concentrator, and run on 4% agarose gel electrophoresis in 1X TBE buffer prestained with SYBR Safe and visualized using the E-Gel Power Snap electrophoresis system.

[0274] Where indicated, primers may incorporate 5' digoxigenin or 5' fluorescein (eg, FAM) or 5' biotin at their corresponding 5' or 3' nucleotides.

[0275] Probe design

[0276] Probes are designed to overlap with the amplified sequence and / or primers (Piepenburg et al.), spanning a GC content between 30% and 70%, and a length between 25nt and 60nt. Where indicated, probes may incorporate 5' digoxigenin, 5' fluorescein (e.g., FAM), reverse 3' dideoxythymidine (reverse_dT), and / or a 3' C3 hydrocarbon spacer (C3) on their corresponding 5' or 3' nucleotides.

[0277] The probe may also have the same sequence as one of the primers, and thus may be referred to as a primer-probe.

[0278] Probe binding was qualitatively assessed by electromobility shift assay (Chen et al.) by incubating 3.2 pmol of probe with 0.2 pmol of complementary duplex in the presence of 32 pmol RecA and 0.3 mM ATPγ-S in 10 μL 1X RecA reaction buffer. If applicable, the molar ratio of probe to target DNA was maintained between 1:1 and 1:16. The molar ratio of RecA to probe was kept constant at 10:1. Optionally, the reaction was supplemented with 5% w / v trehalose ( et al.), 10 mM KCl, 20% w / v PEG 6000 (Teng et al.), and / or ATP (10-fold molar excess compared to ATPγ-S), and / or T4gp32 single-stranded binding protein (SSB) (SSB to RecA molar ratio between 2:1 and 3:1 (Riddles and Lehman)), and / or the ionic liquid 1-ethyl-3-methylimidazolium acetate (Martzy et al.). The reactions were incubated at 37°C for 10 min and run on a 4% agarose gel electrophoresis in 1X TBE buffer prestained to 1X with SYBR Safe and visualized using the E-Gel Power Snap electrophoresis system.

[0279] Example 1 - Effect of Biotin-dCTP Concentration on RPA Yield

[0280] method

[0281] The following 25 μL RPA reactions were incubated at 42° C. for 20 minutes and their fluorescence was read every 30 seconds in the SYBR channel of a real-time quantitative PCR instrument (BioRad): 1× core mix (TwistDx), 1× reaction buffer (TwistDx), 1× basic E-mix (TwistDx), 480 nM primers (each of Fwd_1 (32 nt) and Rev_1 (29 nt)), 100 copies of homologous DNA (Hom_1 (100 bp)), 14 mM magnesium acetate, 0.08 mM dNTP (each), 10%-70% mol / mol biotin-16-dCTP (total dCTP), 1× LAMP fluorescent dye (NEB). According to Munawar et al., the core mix contained GP32SSB, UvsX recombinase, UvsY and polymerase, and the basic E-mix contained creatine phosphate, ATP, creatine kinase, high salt buffer and dH2O.

[0282] result

[0283] A real-time assay was used to evaluate the effect of biotin-16-dCTP incorporation during recombinase polymerase amplification on maximizing the amount of biotin per amplicon. The maximum tolerable amount was found to be approximately 50 molar% of the total dCTP in biotin-16-dCTP. Figure 3 Presented in. Figure 3 A shows real-time RPA amplification of four different conditions, using 0%, 20%, 50% and 70% molar biotin-16-dCTP of total dCTP, n = 2. Fluorescence indicating double-stranded DNA formation was measured every 30 seconds. Figure 3 B shows biotin-16-dCTP compared to Figure 3 % dose response of the area under the curve (AUC) of the amplification curve for A, n=2.

[0284] Example 2 - Effect of biotin-dUTP concentration on RPA yield

[0285] method

[0286] The following 25 μL RPA reactions were incubated at 42°C for 20 min and their fluorescence was read every 30 seconds in the SYBR channel: 1X core mix (TwistDx), 1X reaction buffer (TwistDx), 1X basic E-mix (TwistDx), 480 nM primers (each of Fwd_1 (32 nt) and Rev_1 (29 nt)), 100 copies of homologous DNA (Hom_1 (100 bp)), 14 mM magnesium acetate, 0.08 mM dNTP (each), 10%-70% mol / mol biotin-16-dUTP (of total dUTP), 1X LAMP (NEB).

[0287] result

[0288] A real-time assay was used to evaluate the effect of biotin-16-dUTP incorporation during recombinase polymerase amplification on maximizing the amount of biotin per amplicon. The maximum tolerable amount was found to be approximately 50 molar% of the total dUTP in biotin-16-dUTP. Figure 4 Presented in. Figure 4 A shows real-time RPA amplification of four different conditions, using 0%, 20%, 50% and 70% molar biotin-16-dUTP of total dUTP, n = 2. Fluorescence indicating double-stranded DNA formation was measured every 30 seconds. Figure 4 B shows biotin-16-dUTP compared to Figure 4 % dose response of the area under the curve (AUC) of the amplification curve for A, n=2.

[0289] Example 3 - Effect of the concentrations of biotin-dCTP and biotin-dUTP on RPA yield

[0290] method

[0291] The following 25 μL RPA reactions were incubated at 42°C for 20 min and their fluorescence was read every 30 seconds in the SYBR channel: 1X core mix (TwistDx), 1X reaction buffer (TwistDx), 1X basic E-mix (TwistDx), 480 nM primers (each of Fwd_1 (32 nt) and Rev_1 (29 nt)), 100 copies of homologous DNA (Hom_1 (100 bp)), 14 mM magnesium acetate, 0.08 mM dNTP (each), 10%-70% mol / mol biotin-16-dCTP (of total dCTP) and biotin-16-dUTP (of total dUTP) (each), 1X LAMP (NEB).

[0292] result

[0293] A real-time assay was used to evaluate the effect of biotin-16-dCTP and biotin-16-dUTP incorporation during recombinase polymerase amplification on maximizing the amount of biotin per amplicon. Maximum yields were observed when 50% biotin-16-dUTP and a combination of 40% biotin-16-dCTP and 60% biotin-16-dUTP were used. Results are presented in Figure 5 Presented in. Figure 5 Real-time RPA amplification of six different conditions is shown, using 0%, 50% C and 0% U, 0% U and 50% C, 40% C and 60% U, 50% C and 50% U, and 60% C and 40% U, n = 2. "C" indicates mol / mol of biotin-16-dCTP in total dCTP in a single reaction, and "U" indicates mol / mol of biotin-16-dUTP in total dUTP in a single reaction. Fluorescence indicating double-stranded DNA formation was measured every 30 seconds.

[0294] Example 4 - DNA amplification of target sequences using PCR or RPA

[0295] method

[0296] PCR reaction

[0297] The PCR reaction mixture was collected on ice in a 0.2 mL capped PCR tube and contained 1X Fast Start High Fidelity reaction buffer in a total volume of 25 μL, with 1.8 mM MgCl2, 0.48 μM of each RPA primer, 1 ng DNA template (concentration determined using the Qubit dsDNA High Sense assay kit and Qubit fluorometer), 10% v / v DMSO, 0.44 mM dNTPs (each), 0.44 mM biotin-16-dCTP, 1 unit Fast Start Taq DNA polymerase, and nuclease-free water. The reaction mixture was mixed by brief vortexing and centrifugation. The PCR program was run in an MJ Mini Personal Thermal Cycler (Bio-Rad) and consisted of the following: (1) an initial denaturation step at 95°C for 4 min; (2) 35 cycles of 30 sec at 95°C, 30 sec at 49°C, and 45 sec at 72°C; (3) a final extension step at 72°C for 7 min, followed by quenching at 4°C. PCR products were purified using Zymogen DNAClean & Concentrator and run on 4% agarose gel electrophoresis in 1X TBE buffer prestained with SYBR Safe and visualized using the E-Gel Power Snap electrophoresis system.

[0298] RPA reaction

[0299] The RPA reaction mixture was collected on ice in a 0.2 mL capped PCR tube and contained 1X reaction buffer, 1X E-mix Basic (containing creatine kinase, phosphocreatine and ATP), 1X core reaction mixture, 0.48 μM of each RPA primer, 0.08 mM dNTP (each), 0.08 mM biotin-16-dCTP, 14 mM magnesium acetate, 50 copies of DNA template and nuclease-free water in a total volume of 25 μL. The components of the reaction mixture were mixed with a pipette, and finally the template and magnesium were added to the cap of the tube in separate volumes. The tube was centrifuged, mixed with a pipette on ice, and incubated at 42°C for 20 min in an MJ Mini Personal Thermal cycler (Bio-Rad), then quenched at 4°C. The RPA product was purified using Zymogen DNAClean & Concentrator and run on 4% agarose gel electrophoresis in 1X TBE buffer prestained to 1X with SYBR Safe and visualized using the E-Gel Power Snap electrophoresis system.

[0300] Binding of PCR or RPA amplicons to probes

[0301] The crude PCR or RPA reaction was then further used in Examples 4 to 8.

[0302] PCR or RPA amplicon detection using bridge flocculation

[0303] 25ul crude PCR or RPA reactions were diluted to 100ul with 0.05mg / mL DiagNano PEG-streptavidin silica-coated gold nanoparticles with a diameter of 40nm and a silica shell of 3nm (CD Bioparticles) in 1X PBS, 0.05% Tween-20 and 1% BSA. The solution was incubated at 25°C for 15min. The absorbance of the solution was measured at 520nm and 650nm wavelengths in a flat-bottomed clear 96-well ELISA plate treated with TC (D'Agata et al.).

[0304] result

[0305] As the concentration of PCR or RPA amplification products increases, increased absorbance can be observed.

[0306] Example 5 - Evidence of D-loop formation on agarose gel

[0307] method

[0308] In a 0.2 mL PCR tube, 0.8 pmol homologous DNA (Hom_1 (100 bp), containing 50% mol biotin-16-dCTP), 12.8 pmol forward primer (Fwd_1 (32 nt)) and 128 pmol RecA were mixed in 10 μL 0.3 mM ATPγS, 70 mM Tris-HCl, 10 mM MgCl2 and 5 mM DTT pH 7.6. The mixture was incubated at 37°C for 10 min. 2 μL GelLoading Dye, Purple (6X) (NEB) was added and the samples were run on a 4% w / v agarose gel stained with SYBR Safe (1X) (ThermoFisher) in Tris-borate-EDTA (TBE) buffer (1X) at 28 V for 30 min.

[0309] The above method is based on NEB's protocol "Using RecA and an oligonucleotide to form a stable triple helix" (https: / / international.neb.com / protocols / 0001 / 01 / 01 / using-reca-and-an-oligonucleotide-to-form-a-stable-triple-helix, accessed on January 8, 2022).

[0310] result

[0311] Native agarose gel shows formation of a stable D-loop in the presence of ATPγS, primers, RecA and homologous DNA ( Figure 6 , lane 4). The presence and absence of ATPγS were used for comparison because ATPγS is known to stabilize the D-loop, which is observed as a smear in lane 4. Figure 6 The results of reactions containing different components are shown: lane 1 contains only homologous DNA, lane 2 contains only primers, lane 3 contains homologous DNA, primers and RecA, lane 4 contains homologous DNA, primers, RecA and ATPγS, and lane 5 contains a 100 bp DNA ladder, where the bottom band indicates 100 bp and the band above the bottom band indicates 200 bp.

[0312] Example 6 - Evidence for D-loop formation in ELISA

[0313] method

[0314] The wells of a flat bottom transparent 96-well plate (Greiner) were coated with 5 μg of anti-digoxin polyclonal antibody (Enzo LifeSciences) in 50 μL 14 μM Na2CO3 and 35 μM NaHCO3 pH 9.6 at 4°C for 8 h. The wells were washed three times with 100 μL PBS (1X) and 0.05% v / v Tween-20. The wells were blocked for 30 min at 37°C with 1% w / v BSA in 50 μL PBS (1X) and 0.05% v / v Tween-20. The wells were washed twice with 100 μL PBS (1X) and 0.05% v / v Tween-20, and then washed twice with 100 μL 70 mM Tris-HCl, 10 mM MgCl2 and 0.05% v / v Tween-20 pH 7.6.

[0315] Before removing the wash buffer, in a 0.2 mL PCR tube, 0.1 pmol homologous DNA (Hom_1 (100 bp), containing 50% mol biotin-16-dUTP), 0.1 pmol 5'-digoxigenin reverse primer (Rev_1 (29 nt)) and 10 pmol RecA were mixed in 10 μL 0.3 mM ATPγS, 70 mM Tris-HCl, 10 mM MgCl2 and 5 mM DTT pH 7.6. The mixture was incubated at 37°C for 10 min.

[0316] Remove the last wash buffer from the wells and dilute the RecA reaction to 50 μL with 70mM Tris-HCl, 10mM MgCl2 and 0.05% v / v Tween-20 pH 7.6. Incubate the reaction in the wells at 37°C for 30 min. Wash the wells three times with 100 μL 70mMTris-HCl, 10mM MgCl2 and 0.05% v / v Tween-20 pH 7.6. Incubate the wells with 50 μL 70mM Tris-HCl, 10mM MgCl2 and 0.05% v / v Tween-20 pH 7.6 for 30 min at 37°C. Wash the wells three times with 100 μL 70mM Tris-HCl, 10mM MgCl2 and 0.05% v / v Tween-20 pH 7.6. Detection was accomplished by incubation with 50 μL 0.4 mM TMB, 10% v / v DMSO, 50 mM K2HPO4, 25 mM trisodium citrate and 0.006% w / v H2O2 pH 5.0 at 20°C for 1 min. Color was quenched with 50 uL 0.2 M H2SO4. Absorbance was read at 450 nm.

[0317] result

[0318] A high-throughput D-loop ELISA assay was developed. The use of an ELISA that mimics a lateral flow layout was developed. For this purpose, a 5'-labeled antibody that binds to a primer was immobilized, followed by incubation of the D-loop and detection with a streptavidin-HRP fusion, see schematic diagram Figure 7 A.

[0319] The presence and absence of homologous biotinylated DNA were used for comparison, where a two-fold increase in signal was observed in the presence of DNA, see Figure 7 B. In Figure 7 In B, the left bar shows the absorbance of the experiment using homologous DNA containing 50% mol biotin-16-dUTP, while the right bar shows the absorbance of the experiment in which the homologous DNA was replaced by H2O.

[0320] Example 7 - Evidence for D-loop formation in lateral flow assays

[0321] method

[0322] In a 0.2 mL PCR tube, 0.8 pmol homologous DNA ((Hom_1 (100 bp), containing 50% mol biotin-16-dCTP), 12.8 pmol 5'FAM reverse primer (Rev_1 (29 nt)) and 128 pmol RecA were mixed in 10 μL 0.3 mM ATPγS, 70 mM Tris-HCl, 10 mM MgCl2 and 5 mM DTT pH 7.6. The mixture was incubated at 37°C for 10 min. The mixture was diluted to 150 uL in a 1.5 mL Eppendorf tube with PCRD extraction buffer (Abington Health). PCRD FLEX lateral flow strips (Abington Health) coated with anti-digoxigenin, anti-fluorescein and anti-streptavidin were immersed in the solution, incubated at 25°C, removed after 10 min, and visually inspected.

[0323] result

[0324] The possibility of using stable D-loop formation to detect DNA on lateral flow strips was evaluated. The concept of the assay is presented in Figure 8 Diagram in A. The presence and absence of ATPγS were used for comparison because ATPγS is known to stabilize the D-loop, which is observed as a band at the FAM binding site in the lateral flow strip, see Figure 8 B. In Figure 8 In B, "D" indicates a digoxigenin binding site, "F" indicates a FAM binding site, and "S" indicates a streptavidin binding site. The top bar shows the results of an experiment using ATPγS, and the bottom bar shows the results of an experiment not using ATPγS.

[0325] Example 8 - Effect of probe design on lateral flow assays

[0326] method

[0327] In a 0.2 mL PCR tube, 0.2 pmol homologous DNA (Hom_1 (100 bp), containing 50% mol biotin-16-dUTP), 0.2 pmol probe (5' digoxigenin-Rev_1 (29 nt), 5' digoxigenin-probe_1 (25 nt)-3' reverse_dT or 5' digoxigenin-probe_2 (60 nt)-3' C3 spacer) and 2 pmol RecA were mixed in 10 μL 0.3 mM ATPγS, 70 mM Tris-HCl, 10 mM MgCl2 and 5 mM DTT pH 7.6. The mixture was incubated at 37°C for 10 min. The mixture was diluted to 150 uL in a 1.5 mL Eppendorf tube with PCRD extraction buffer (Abington Health). PCRD FLEX lateral flow strips (Abington Health) coated with anti-digoxigenin, anti-fluorescein and anti-streptavidin were Health) were immersed in the solution, incubated at 25°C, taken out after 10 min, and visually inspected.

[0328] The optional conditions for the reaction were no additives or one of the following during the reaction: 6 pmol T4gp32 single-chain binding protein; 5% w / v trehalose, 20% w / v PEG 6000, 100 mM KCl; 80 mM 1-ethyl-3-methylimidazolium acetate (Iolitec); or a combination of the three.

[0329] result

[0330] The stability of D-loop formation in various probe designs was evaluated as a function of their design, which refers to binding to the ends of biotinylated homologous DNA (5' digoxigenin-Rev_1 (29 nt), Fig. 9 A and Fig. 9 B) or middle (5' digoxigenin-probe_1(25nt)-3' reverse_dT, Fig. 9 C and Fig. 9 D, or 5' digoxigenin-probe_2 (60nt)-3'C3 spacer, Fig. 9 E and Fig. 9F). In addition, several conditions known to stabilize the D-loop were tried, since D-loops formed at the ends (i.e., plectonemic joints) are more stable than those formed in the middle of a DNA duplex (i.e., parallel sliding joints). These conditions were the presence of single-stranded binding proteins, PEG, KCl, trehalose, the ionic liquid 1-ethyl-3-methylimidazolium acetate, and combinations thereof. The results showed that the end probes were able to bind regardless of the conditions, while the binding of the middle binding probes varied with their length and additives. The best results were achieved with a 60nt probe (5' digoxigenin-probe_2 (60nt)-3'C3 spacer) that partially overlapped the primer sequences Fwd_1 and Rev_1 in the presence of ionic liquids, see Fig. 9 F. This is surprising since the use of ionic liquids to improve D-ring stabilization has not been described before.

[0331] Fig. 9 A. Fig. 9 C and Fig. 9 E shows a schematic diagram of the detection concept of different probes. Fig. 9 B. Fig. 9 D and Fig. 9 F shows the lateral flow assay Fig. 9 A. Fig. 9 C and Fig. 9 Results of the corresponding concepts of E. "D" indicates digoxigenin binding site, "F" indicates FAM binding site, and "S" indicates streptavidin binding site. Bar 1 indicates no additives, bar 2 indicates added single-chain binding protein, bar 3 indicates added trehalose, PEG 6000 and KCl, bar 4 indicates added ionic liquid 1-ethyl-3-methylimidazolium acetate, and bar 5 indicates added single-chain binding protein, trehalose, PEG, KCl and ionic liquid 1-ethyl-3-methylimidazolium acetate.

[0332] Example 9 - Specificity of primer-probes and probes with and without ATP-γ-S compared to hapten-based strategies

[0333] method

[0334] The following 25 μL reaction was incubated at 37°C for 20 min: 1X Core Mix (TwistDx), 1X Reaction Buffer (TwistDx), 1X Basic E-Mix (TwistDx), 450 nM primers (each), 20,000 copies of DNA (Hom_1 (100 bp) or Non_1 (140 bp)), 14 mM magnesium acetate, 0.08 mM dNTPs (each), 50% mol / mol biotin-16-dUTP (of total dUTP). Primers were (1) unmodified primers (Fwd_1 (32 nt) and Rev_1 (29 nt)), (2) a mixture of a 5'biotin forward primer (5'biotin-Fwd_1 (32 nt)) and a 5'-digoxigenin reverse primer (5'digoxigenin-Rev_1 (29 nt)), or (3) unmodified primers targeting sequences different from (1) and (2) (Fwd_2 (30 nt) and Rev_2 (30 nt)). The reaction was purified using the GeneJET PCR purification kit (ThermoFisher) and quantified using the Qubit High-Sense DNA kit (ThermoFisher).

[0335] DNA amplified by the above reaction was added at 50 ng to the following 10 uL reaction: 1.28 pmol probe (5' digoxigenin-Rev_1 (29 nt) or 5' digoxigenin-probe_2 (60 nt)-C3 spacer), 128 pmol RecA, 0.3 mM ATPγS, 70 mM Tris-HCl, 10 mM MgCl2, and 5 mM DTT pH 7.6. For different conditions, ATPγS was replaced with 3 mM ATP or 1x basic E-mix from the TwistDx Liquid Basic kit, which contained 50 mM creatine phosphate, 3 mM ATP, and 100 ng / uL creatine kinase. For yet another condition, ATPγS was replaced with a combination of ATPγS and ATP and 1x basic E-mix, where the molar ratio of ATP to ATPγS was 10:1.

[0336] Probe binding was quantitatively assessed using an ELISA-based assay. A flat-bottomed transparent 96-well ELISA plate treated with anti-digoxigenin antibody was coated with TC by incubating the wells with 50 μg of antibody in 14 mM Na2CO3 and 35 mM NaHCO3 pH 9.6 at 37°C for 30 min. The wells were washed three times with 1X wash buffer (70 mM Tris-HCl, 10 mM MgCl2, 0.05% v / v Tween-20, pH 7.0-8.0). The wells were blocked at 37°C with blocking buffer (70 mM Tris-HCl, 10 mM MgCl2, 1% w / v BSA, pH 7.0-8.0) for 30 min and washed four times. The RPA amplicon pre-incubated with the probe between 1 fmol and 1 pmol was incubated at 37°C for 30 min and washed three times. Incubate 1 μg / mL enhanced streptavidin-HRP from Kementec in wash buffer at 37°C for 30 min and wash three times. Add 50 μl of 0.04 mg / mL TMB freshly prepared in 50 mM K2HPO4, 20 mM sodium citrate, 1% v / v DMSO, and 0.06% w / w H2O2 to the wells and incubate for 2-10 min at 25°C. Add 50 μL 0.2M H2SO4 to stop color development and measure absorbance at 450 nm with a TECAN multi-well plate reader.

[0337] result

[0338] The extent of sequence-specific binding of a 29nt primer-probe (5' Digoxigenin-Rev_1 (29nt)) and a 60nt probe (5' Digoxigenin-Probe_2 (60nt)-C3 Spacer) to the middle of the amplicon on templates amplified and purified by RPA using 50% biotin-16-dUTP was evaluated. RPA amplifications were compared with hapten-labeled primers (5' Biotin-Fwd_1 (32nt)) and 5' Digoxigenin-Rev_1 (29nt)). The effects of replacing ATP-γ-S with limiting amounts of ATP and ATP with an ATP regeneration system (phosphocreatine and creatine kinase) and its combination with ATP-γ-S were plotted.

[0339] Fig.10A shows the specificity of primer-probe (helix-wound ligation) D-loop detection using ELISA. It shows the results of experiments in which the DNA template Hom_1 (100 bp) was amplified using Fwd_1 (32 nt) and Rev_1 (29 nt), resulting in the reference "Ho", or in which the DNA template Non_1 (140 bp) was amplified using Fwd_2 (30 nt) and Rev_2 (30 nt), resulting in the reference "No". The amplified DNA was subjected to primer-probe (5' digoxigenin-Rev_1 (29 nt), indicated by "Pri") and recombinase incubation using different ATP / ATPγS inputs. The different ATP / ATPγS inputs are: ATP regeneration via ATP with creatine kinase and phosphocreatine (1× basic E-mix), indicated by “rA”; ATPγS alone, indicated by “Aγ”; ATP alone, indicated by “A”; a combination of rA and Aγ, indicated by “rA / Aγ”; a combination of A and Aγ, indicated by “A / Aγ”. The resulting D-loops were then detected using ELISA.

[0340] Since the primer-probe is not complementary to the non-homologous DNA template No, a D-loop should not form, resulting in a signal that can be considered a negative signal. In contrast, the primer-probe is complementary to the DNA template Ho, resulting in a D-loop and a positive signal. Fig.10 It can be observed in A that for each ATP / ATPγS input condition, the positive signal is higher than the negative signal, confirming the presence of the D-loop. The greater difference between the negative and positive signals for the corresponding ATP / ATPγS input condition can be considered as a greater specificity of the probe for the corresponding condition. Fig.10 In A, the greatest specificity was observed for the (Pri-)rA / Aγ condition, while the lowest specificity was observed for the (Pri-)A condition. In general, higher specificity was observed when ATPγS was used.

[0341] Fig.10B shows the specificity of probe-based (parallel slide ligation) D-loop detection using ELISA. It shows the results of experiments in which the DNA template Hom_1 (100 bp) was amplified using Fwd_1 (32 nt) and Rev_1 (29 nt), resulting in the reference "Ho", or in which the DNA template Non_1 (140 bp) was amplified using Fwd_2 (30 nt) and Rev_2 (30 nt), resulting in the reference "No". The amplified DNA was subjected to probe (5' digoxigenin-probe_2 (60 nt)-C3 spacer, indicated by "Pro") and recombinase incubation using different ATP / ATPγS inputs. The different ATP / ATPγS inputs are: ATP regeneration via ATP with creatine kinase and phosphocreatine (1× basic E-mix), indicated by “rA”; ATPγS alone, indicated by “Aγ”; ATP alone, indicated by “A”; a combination of rA and Aγ, indicated by “rA / Aγ”; a combination of A and Aγ, indicated by “A / Aγ”. The resulting D-loops were then detected using ELISA.

[0342] Since the probe is not complementary to the non-homologous DNA template No, a D-loop should not form, resulting in a signal that can be considered a negative signal. In contrast, the probe is complementary to the DNA template Ho, resulting in a D-loop and a positive signal. Fig.10 It can be observed in B that for each ATP / ATPγS input condition, the positive signal is higher than the negative signal, confirming the presence of the D-loop. The greater difference between the negative and positive signals for the corresponding ATP / ATPγS input condition can be considered as a greater specificity of the probe for the corresponding condition. Fig.10 In B, the greatest specificity is observed for the (Pro-)A / Aγ condition, while the lowest specificity is observed for the (Pro-)rA condition. In general, higher specificity is observed when ATPγS is used.

[0343] In addition, comparison Fig.10 A and Fig.10 B, it can be observed that the probe-based D-loop detection has greater specificity than the primer-probe-based D-loop detection. This is surprising because D-loops formed at the ends (i.e., helical winding junctions) are more stable than D-loops formed in the middle of a DNA duplex (i.e., parallel sliding junctions).

[0344] Fig.10 C shows a comparison of the specificity of D-loop detection using ELISA with conventional double hapten labeled RPA amplicons. Conditions Pro-No-A / Aγ, Pro-Ho-A / Aγ, Pri-No-A / Aγ, and Pri-Ho-A / Aγ were taken from Fig.10 B and Fig.10A. In addition, two conventional double hapten-labeled RPA amplicons were generated as follows: 5' biotin-Fwd_1 (32 nt) and 5' digoxigenin-Rev_1 (29 nt) were used to amplify the DNA template Hom_1 (100 bp) and the DNA template Non_1 (140 bp), respectively, to generate the reference "5'DIG-5'bio-Ho" (RPA specific amplification product) and "5'DIG-5'bio-No" (i.e., RPA nonspecific amplification product), respectively. Fig.10 A and Fig.10 The products were detected by ELISA under the same conditions as described in B.

[0345] It can be observed that D-loop detection according to the present invention, especially when a (stable) parallel sliding junction is formed, produces an improved signal compared to detection of RPA-amplified DNA by conventional double hapten labeling, where detection of RPA-amplified DNA by conventional double hapten labeling produces a lower relative signal (i.e., the signal of 5'DIG-5'bio-Ho minus the signal of 5'DIG-5'bio-No).

[0346] Example 10 - RPA amplicons can be added directly to an immobilized D-loop assay coupled to a RecA probe, which is highly reproducible and specific.

[0347] RPA reaction, RecA reaction and ELISA (evidence of D-loop formation in ELISA) were performed as described in Example 6. In short, Fwd_1 and Rev_1 were used to amplify Hom_1 with 30% biotin-16-dUTP in the RPA reaction, and then the crude RPA reaction was serially diluted in deionized water. The serial dilutions were added to the RecA reaction with 0.3mM ATPγS and probe_2 in triplicate with a volume of 1 μL, and then added to the ELISA in triplicate (Pro-HO-Aγ, crude, [dilution factor]). As a negative control, the input amplicon in the RecA reaction was Non_1 (Pro-No-Aγ) with 30% biotin-16-dUTP of 1ng amplification and purification in triplicate. As a positive control for ELISA, the input amplicon in the ELISA was 5'DIG-5'bio-Ho (5'DIG-5'bio-Ho) of 1ng amplification and purification in triplicate. As a positive control for the RecA reaction, the input amplicon in RecA was 1 ng of amplified and purified Hom_1 (Pro-Ho-Aγ) with 30% biotin-16-dUTP in triplicate runs. As a specificity test, Non_1 was amplified in RPA with 30% biotin-16-dUTP and the crude RPA reaction was serially diluted in deionized water. The serial dilutions were added in 1 μL volumes to triplicate RecA reactions with 0.3 mM ATPγS and Probe_2 and subsequently added to the ELISA in triplicate (Pro-No-Aγ, crude, [dilution factor]). The results are in Fig.11 Shown in.

[0348] Comparison between serial dilutions of Hom_1 and Non_1 amplicons in crude RPA reactions shows that the specificity and reproducibility of the D-loop based detection method are preserved even after adding the crude RPA product directly to the RecA reaction. Comparison of different dilutions within Hom_1 also shows that the signal is proportional to the amount of product added, again indicating that the detected material is the RPA amplicon, and that the specificity of the detection is preserved when the same dilutions are compared between Hom_1 and Non_1 amplicons. This example shows that the method according to the invention can be performed as a one-pot reaction.

[0349] Example 11 - DrRecA shows strand exchange.

[0350] A real-time fluorescence assay for recombinase chain exchange activity was developed. In short, 10 pmol of dsDNA annealed from 5'FAM-Fwd_1 and AntiFwd_1-3'BHQ1 was added to the RecA reaction with 10 pmol FWD_1. When the recombinase and ATP source (E-mix, ATP regeneration system) were added, Fwd_1 replaced its FAM-labeled analog close to BHQ1 in the dsDNA, resulting in an increase in fluorescence because chain exchange during D-loop formation occurred in the presence of the recombinase and ATP source. The products can also be run on agarose gels, where the presence of chain exchange products can be observed as bands with brighter intensity due to the fluorescence of the released FAM. Interestingly, the chain exchange activity of the RecA enzyme (DrRecA) from Deinococcus radiodurans was detected and was independent of the presence of an ATP source. This was evident when comparing the fluorescence traces of conditions with and without an ATP source and the corresponding conditions in an agarose gel. As a negative control for fluorescence assay and agarose gel, conditions without recombinase ( Fig.12 As a positive control for the agarose gel, conditions containing E. coli RecA and an ATP source ( Fig.12 The results of real-time fluorescence assay are shown in Fig.12 A. The results of the agarose gel experiment are shown in Fig.12 In B.

[0351] Example 12 - Bridge flocculation allows visual detection of biotinylated DNA.

[0352] The streptavidin-coated magnetic beads (Cell Signaling Technology) were pulled down with a magnet and washed once with wash buffer (70 mM Tris-HCl, 10 mM MgCl2, 0.05% v / v Tween-20, pH 8.0). 10 μL of the purified RPA amplicon of Hom_1 with 30% biotin-dUTP was mixed with streptavidin-coated magnetic beads (Cell signaling technology, product number 5947S) in a 1:1 volume and incubated at 37°C for 5 min. After washing the beads twice with wash buffer, 10 μL of 1 μg / mL enhanced streptavidin-horseradish peroxidase (Kementec) in wash buffer was added and incubated at 37°C for 5 min. After washing the beads twice in wash buffer, a volume of 300 μL of 0.1 mg / mL TMB and 0.6% H2O2 in phosphate-citrate buffer at pH 5.0 was added and incubated for 1 min at room temperature. As a negative control, the purified product of the RPA reaction without the template Hom_1 was used (ie, no template control).

[0353] The significant difference in color development between samples treated with purified RPA product with and without template DNA indicates that this method can be used to detect biotinylated RPA amplicons by eye after RPA. The results are shown in Fig.13 Middle (left: no template control; right: template positive).

[0354] Example 13 - Stable D-loop formation by EcRecA and DrRecA.

[0355] RPA reactions, RecA reactions, and ELISA were performed as described in Example 9. Specifically, DNA template Hom_1 (100 bp) was amplified via RPA using Fwd_1 and Rev_1. The amplified DNA ("Ho") was subjected to probe (5' digoxigenin-probe_2-C3 spacer) ("Pro-Ho") and recombinase incubation using different ATP inputs. Equimolar amounts of Pro-Ho probe and Ho template (300 fmol each) were used. Different ATP inputs were: 10 mM Tris-HCl pH 8.0 (no ATP) 1mM ATP (A), 10mM dATP (dA), 1mM ATPγS (Aγ) and ATP regeneration system (rA) (2.5mM ATP, 300ng / μL creatine kinase, 50mM phosphocreatine). The final concentrations of Escherichia coli RecA (EcRecA) and Deinococcus radiodurans RecA (DrRecA) were 0.2μg / μL and 0.6μg / μL, respectively. The resulting stable D-loop was then detected using ELISA. The results are shown in Fig.14 A (EcRecA) and Fig.14 B (DrRecA).

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[0375] -D’Agata,R.;Palladino,P.;Spoto,G.Streptavidin-Coated GoldNanoparticles:Critical Role of Oligonucleotides on Stability and FractalAggregation.Beilstein J.Nanotechnol.2017,8,1-11.doi:10.3762 / bjnano.8.1.

Claims

1. A method for determining the presence or absence of a target deoxyribonucleic acid (DNA) sequence in a sample, the method comprising: a. contacting the sample with: a first primer and a second primer for amplifying the target DNA sequence; Recombinase; DNA-dependent DNA polymerase; A deoxynucleoside triphosphate (dNTP) monomer containing a first label; and An oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a second label; b. amplifying the target DNA sequence to produce a nucleic acid amplification product; and c. detecting the nucleic acid amplification product, wherein the nucleic acid amplification product comprises a D-loop, the D-loop comprises triple-stranded DNA, the probe and the recombinase, wherein detecting the nucleic acid amplification product comprises detecting the probe, Contacting the sample with the recombinase and / or the probe may be performed before, during or after step b. amplifying the target DNA sequence.

2. The method according to claim 1, wherein detecting the nucleic acid amplification product in step c comprises binding one or more first labels of the dNTP monomers of the nucleic acid amplification product to a first binding agent, and binding the second label of the probe bound to the nucleic acid amplification product to a second binding agent, preferably, wherein the first label is not bound to the second binding agent, and the second label is not bound to the first binding agent, and / or wherein the first binding agent is attached to a solid support.

3. The method according to claim 1 or 2, wherein contacting the sample with a dNTP monomer containing a first label comprises contacting the sample with a mixture comprising: 30% to 70% deoxycytidine triphosphate (dCTP) containing a first label, 30% to 70% deoxyadenosine triphosphate (dATP) containing a first label, 30% to 70% deoxyguanosine triphosphate (dGTP) containing a first label, 30% to 70% deoxythymidine triphosphate (dTTP) containing a first label and / or 30% to 70% deoxyuridine triphosphate (dUTP) containing a first label, each based on the total molar number of dNTPs of its base type, preferably wherein the first label comprises biotin.

4. The method according to any one of claims 1 to 3, further comprising contacting the sample with: a third primer and a fourth primer for amplifying a second target DNA sequence; and a second oligonucleotide probe comprising a nucleic acid sequence complementary to the second target DNA sequence and a third label.

5. The method according to claim 4, wherein detecting the nucleic acid amplification product in step c also includes combining the third label of the second probe bound to the nucleic acid amplification product with a third binding agent, preferably wherein the first label and the third label are not bound to the second binding agent, the second label and the third label are not bound to the first binding agent, and the first label and the second label are not bound to the third binding agent.

6. A method for determining the presence or absence of a target DNA sequence in a sample, the method comprising: a. Contact the sample with: a first primer and a second primer for amplifying the target DNA sequence, wherein the second primer contains a second label and hybridizes to a first strand of DNA of the target DNA sequence; Recombinase; DNA-dependent DNA polymerase; and an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label; b. amplifying the target DNA sequence to produce a nucleic acid amplification product; as well as c. detecting the nucleic acid amplification product, wherein the nucleic acid amplification product comprises a D-loop, the D-loop comprises triple-stranded DNA, the probe and the recombinase, wherein detecting the nucleic acid amplification product comprises detecting the probe, Contacting the sample with the recombinase and / or the probe may be performed before, during or after step b. amplifying the target DNA sequence. The method of claim 6 , wherein the probe comprises at least two nucleotides containing a first label.

8. The method according to claim 6 or 7, wherein detecting the nucleic acid amplification product in step c comprises binding one or more first labels of the nucleotides of the probe to a first binding agent, and binding the second label of the second primer bound to the nucleic acid amplification product to a second binding agent, preferably, wherein the first label is not bound to the second binding agent, and the second label is not bound to the first binding agent, and / or wherein the second binding agent is attached to a solid support.

9. The method according to any one of claims 6 to 8, wherein the probe comprises 10% to 90%, preferably 20% to 80%, of nucleotides containing the first label based on the total number of nucleotides of the probe.

10. The method according to any one of claims 6 to 9, further comprising contacting the sample with: a third primer and a fourth primer for amplifying a second target DNA sequence, wherein the fourth primer contains a third label and hybridizes with the first strand of DNA of the second target DNA sequence and a second oligonucleotide probe, wherein the second oligonucleotide probe comprises a nucleic acid sequence complementary to the second target DNA sequence and one or more nucleotides containing the first label.

11. The method according to claim 10, wherein detecting the nucleic acid amplification product in step c also includes binding the third label of the fourth primer bound to the nucleic acid amplification product to a third binding agent, preferably wherein the first label and the third label are not bound to the second binding agent, the second label and the third label are not bound to the first binding agent, and the first label and the second label are not bound to the third binding agent.

12. The method according to any one of claims 1 to 11, wherein the probe comprises a polymerase blocking group, preferably wherein the blocking group comprises a hydrocarbon spacer, an inverted deoxynucleotide, a phosphorylation, a primary amine or a dideoxynucleotide.

13. The method of claim 12, wherein the blocking group is located at the 3' end of the probe, wherein the end is 0 to 10 nucleotides, inclusive, of the 3' terminal nucleotide of the probe.

14. The method according to any one of claims 1 to 13, wherein the first label comprises or is biotin or a derivative thereof, and the second label is not or does not comprise biotin or a derivative thereof, preferably the dNTP monomer comprising the first label comprises biotin-labeled dCTP, biotin-labeled dUTP, biotin-labeled dGTP, biotin-labeled dTTP and / or biotin-labeled dATP, or preferably the nucleotide comprising the first label comprises biotin-labeled cytosine nucleotide, biotin-labeled adenine nucleotide, biotin-labeled guanine nucleotide, biotin-labeled thymidine nucleotide and / or biotin-labeled uracil nucleotide.

15. The method according to any one of claims 1 to 14, wherein the first and / or second label is a hapten or a covalently attached label.

16. The method according to any one of claims 1 to 15, wherein the second label is a hapten, preferably fluorescein, its derivatives such as 6-FAM and FITC, digoxigenin, Texas red, dansyl, Cascade blue or dinitrophenol.

17. according to the method described in any one of claims 1 to 16, wherein the second label is located at the 5' end of the probe, at the 3' end of the probe or in the middle of the probe, wherein the end is 0 to 10 nucleotides of the 5' or 3' terminal nucleotide of the probe, respectively, including the terminal nucleotide, and the middle is 0 to 10 nucleotides of the central nucleotide of the probe, including the central nucleotide.

18. The method according to any one of claims 1 to 17, wherein the target DNA sequence is amplified by performing an isothermal amplification reaction.

19. The method of claim 18, wherein the isothermal amplification reaction is a recombinase polymerase amplification (RPA) reaction.

20. The method according to any one of claims 1 to 19, wherein the sample is contacted with the probe during step b or after step b, preferably wherein the sample is not contacted with the probe before step b.

21. The method of claim 20, wherein the sample is contacted with the probe after step b, and the sample is contacted with an additional amount of a recombinase; or wherein the sample is contacted with a recombinase only after step b.

22. The method according to any one of claims 1 to 21, wherein the recombinase is a recombinase selected from the group consisting of: RecA, DrRecA, Rad51, RadA or UvsX recombinase or a homologue thereof.

23. The method according to any one of claims 1 to 22, further comprising contacting the sample with a component for modulating binding of the recombinase or labeled probe to the nucleic acid amplification product.

24. The method according to any one of claims 1 to 23, further comprising adding an ATP analogue, preferably a non-hydrolyzable or hydrolysis-resistant ATP analogue, before, during or after amplifying the target DNA sequence.

25. The method according to any one of claims 1 to 24, further comprising contacting the sample with a double-stranded DNA destabilizer and / or a triple-stranded DNA stabilizer, preferably wherein the sample comprises the nucleic acid amplification product or the isolated nucleic acid amplification product.

26. The method according to any one of claims 1 to 25, further comprising contacting the sample with an RNA-dependent DNA polymerase to produce the target DNA sequence before step a.

27. The method according to any one of claims 1 to 26, wherein the dNTP monomer containing a first label or the nucleotide containing a first label is a biotin-labeled dNTP monomer or a biotin-labeled nucleotide, respectively, preferably wherein the biotin is covalently linked to the dNTP monomer or the nucleotide via a linker of 11 to 18 atoms, more preferably 11, 14, 16 or 18 atoms, most preferably 16 atoms, respectively.

28. The method according to claim 27, wherein the biotin-labeled dNTP monomer comprises biotin-labeled dCTP and / or biotin-labeled dUTP, preferably biotin-16-dCTP and / or biotin-16-dUTP, or wherein the biotin-labeled nucleotide comprises biotin-labeled cytosine and / or biotin-labeled uracil, preferably biotin-16-cytosine and / or biotin-16-uracil.

29. The method according to any one of claims 1 to 28, wherein the probe comprises 30% to 70% dCTP and / or dGTP nucleotides based on the total number of nucleotides in the probe, optionally wherein the nucleotides contain a first label.

30. The method according to any one of claims 1 to 29, wherein the nucleic acid sequence in the probe that is complementary to the target DNA sequence or the nucleic acid sequence in the target DNA sequence to which the probe hybridizes does not overlap with the nucleic acid sequence to which the first primer or the second primer hybridizes, or wherein the nucleic acid sequence in the probe that is complementary to the target DNA sequence or the nucleic acid sequence in the target DNA sequence to which the probe hybridizes partially overlaps with the nucleic acid sequence to which the first primer or the second primer hybridizes, preferably wherein the partial overlap is 1 to 10 nucleotides.

31. The method according to any one of claims 1 to 30, wherein the probe comprises a partial nucleic acid sequence or a complete nucleic acid sequence of one of the primers used for amplification, preferably, wherein the partial sequence comprises at least 1% of the complete nucleic acid sequence of one of the primers used for amplification or a first primer used for amplification.

32. The method of any one of claims 1 to 31, wherein contacting the sample comprises contacting the sample with a lesser amount of the probe than the amount of the first primer and / or the second primer on a molar basis, preferably wherein the molar ratio of each of the first primer and the second primer to the probe is from 1:1 to 100:

1.

33. The method of any one of claims 1 to 32, wherein the first binding agent comprises avidin, streptavidin, neutravidin, an anti-biotin antibody or a binding agent specific for biotin and / or wherein the second binding agent comprises an antibody.

34. The method of any one of claims 1 to 33, wherein the first binding agent comprises nanoparticles, colored (nano)particles, gold (nano)particles, latex, fluorescent compounds, luminescent compounds, or magnetic compounds.

35. The method according to any one of claims 1 to 34, wherein detecting the nucleic acid amplification product is performed during amplification of the target DNA sequence, preferably, wherein the probe, the first label or the second label comprises a fluorescent compound.

36. The method of any one of claims 1 to 35, wherein detecting the nucleic acid amplification product comprises using a lateral flow test, a lateral flow device, an enzyme-linked immunosorbent assay, a magnetic immunoassay, or a flocculation assay.

37. The method of claim 36, comprising: a. providing a lateral flow device comprising a first binding agent capable of binding to said first marker and a second binding agent capable of binding to said second marker, preferably wherein said second binding agent is immobilized, preferably immobilized on said lateral flow device; b. diluting the nucleic acid amplification product, preferably using a buffer having a pH of 6 to 8, preferably about 7; c. applying the diluted nucleic acid amplification product to a lateral flow test strip of the lateral flow device; and d. Detecting the product, if present, on the test zone of the lateral flow strip.

38. The method of claim 37, wherein the buffer comprises a non-ionic detergent, preferably a non-denaturing non-ionic detergent.

39. The method according to claim 36 comprises combining the nucleic acid amplification product and the first binding agent, wherein the product and the first binding agent are capable of forming a visually detectable complex, wherein the complex is formed by flocculation, preferably by flocculation of the first binding agent, and more preferably by flocculation of the first binding agent by the nucleic acid amplification product.

40. The method of claim 39, wherein the presence or relative amount of the nucleic acid amplification product is determined by the presence or increased flocculation compared to flocculation observed in the absence of the nucleic acid amplification product or at a lower amount or concentration of the nucleic acid amplification product.

41. A method according to claim 39 or 40, wherein the first binding agent comprises avidin, streptavidin, neutravidin, anti-biotin antibody or a binding agent specific for biotin bound to one or more colored nanoparticles, silver nanoparticles or gold nanoparticles, preferably, wherein the nanoparticles have a diameter of 5nm to 50nm and / or have a silica shell with a diameter of 2nm to 4nm.

42. The method according to any one of claims 39 to 41, further comprising diluting the complex with a buffer having a pH of 6 to 8, preferably about 7, preferably wherein the buffer comprises a non-ionic detergent, preferably a non-denaturing non-ionic detergent.

43. An ex vivo method for diagnosing a disease or condition, preferably an infectious disease, in a subject, comprising: a. Determining the presence or absence of a target DNA sequence associated with a disease or disorder according to any one of claims 1 to 42 in a sample obtained from the subject, wherein the presence is indicative of a diagnosis of the disease or disorder in the subject.

44. A method for detecting a nucleic acid amplification product comprising a target DNA sequence, the method comprising: a. contacting the nucleic acid amplification product with a recombinase and an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a label, thereby forming a D-loop comprising triple-stranded DNA, the probe and the recombinase in the nucleic acid amplification product; and b. Detecting the nucleic acid amplification product, including detecting the probe.

45. The method of claim 44, wherein detecting the nucleic acid amplification product comprises binding the label of the probe bound to the nucleic acid amplification product to a binding agent, preferably wherein the binding agent is attached to a solid support, more preferably wherein detecting the nucleic acid amplification product comprises using a lateral flow test, a lateral flow device, an enzyme-linked immunosorbent assay, a magnetic immunoassay, or a flocculation assay.

46. ​​The method according to any one of claims 1 to 45, wherein the method is nuclease-free, preferably nuclease-free and protease-free.

47. A composition, said composition being free of nuclease, said composition comprising: A dNTP monomer containing a first label and an oligonucleotide probe containing a second label; or A first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and is capable of hybridizing to a first strand of DNA of the target DNA sequence; And an oligonucleotide probe, which comprises a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label, preferably, wherein the probe is capable of binding or hybridizing to the first strand or the second strand of DNA of the target DNA sequence, the second strand being complementary to the first strand.

48. A lateral flow device, comprising a lateral flow strip, the lateral flow strip comprising: - sample application zone; - a reagent zone downstream of the sample application zone and in fluid communication with the sample application zone, wherein the reagent zone comprises a non-immobilized first binding agent bound to a first label; - a test zone downstream of and in fluid communication with the reagent zone, wherein the test zone comprises an immobilized second binding agent bound to a second label; and - a control zone downstream of said test zone and in fluid communication with said test zone.

49. The device according to claim 48, wherein the reagent zone comprises a recombinase and / or a dried RPA composition for amplifying a target DNA sequence, preferably comprises a composition according to claim 47.

50. Apparatus according to claim 48 or 49, for use in a method according to any one of claims 1 to 46.

51. A kit of parts, the kit being free of nucleases, the kit comprising: a. Optionally, a first primer and a second primer for amplifying a target DNA sequence; b. Recombinase; c.DNA-dependent DNA polymerase; d. a dNTP monomer containing a first label; and e. an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and a second label; and optionally f. a lateral flow device or a device according to claim 48 or 49; and optionally g. Instructions for carrying out the method according to any one of claims 1 to 46.

52. A kit of parts, the kit being free of nuclease, the kit comprising: a. a first primer and a second primer for amplifying a target DNA sequence, wherein the second primer contains a second label and hybridizes to a first strand of DNA of the target DNA sequence; b. Recombinase; c.DNA-dependent DNA polymerase; and d. an oligonucleotide probe comprising a nucleic acid sequence complementary to the target DNA sequence and one or more nucleotides containing a first label, preferably wherein the probe hybridizes to a first strand or a second strand of DNA of the target DNA sequence, the second strand being complementary to the first strand; and optionally e. a lateral flow device or a device according to claim 48 or 49; and optionally f. Instructions for carrying out the method according to any one of claims 1 to 46.

53. A composition according to claim 47 or a kit of parts according to claim 51 or 52, further being protease-free and / or for use in a method according to any one of claims 1 to 46.

54. Use of an apparatus according to claim 48 or 49 or a kit of parts according to claim 51 or 52 for determining the presence or absence of a pathogen, preferably a human pathogen, an animal pathogen or a plant pathogen, or a target DNA sequence associated with a disease or disorder or the onset thereof.

55. The method of claim 43 or the use of claim 54, wherein the disease or condition is cancer, an autoimmune disease or condition, a genetic disease or condition, pain, a respiratory disease or condition, a cardiovascular disease or condition, an infectious disease or condition, a thyroid disease or condition, a skin disease or condition, diabetes, obesity, a kidney disease or condition, a gastrointestinal disease or condition, a neurodegenerative disease or condition, a musculoskeletal disease or condition, a metabolic disease or condition, or a psychiatric disorder, preferably cancer, an autoimmune disease or condition, or an infectious disease or condition.

56. The use according to claim 54, wherein the target DNA sequence associated with a disease or disorder or the onset thereof comprises a DNA sequence expressing a tumor antigen, a tumor marker or a tumor suppressor.

57. Use of the device according to claim 48 or 49 or the kit according to claim 51 or 52 for determining the presence or absence of a target DNA sequence in a diagnostic sample, a veterinary sample, a pharmaceutical sample, a cosmetic sample, a food sample, a beverage sample, a feed sample, an agricultural sample, a soil sample, an air sample, a water sample, a waste sample, an effluent sample or a sewage sample.

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