Method, primer set, and kit for simultaneously amplifying a plurality of target nucleic acid sequences in a sample

Abstract

The invention relates to a method for simultaneously amplifying a plurality of target nucleic acid sequences in a sample, said method having the step of: (S300) amplifying the target nucleic acid sequences by means of an isothermal strand displacement amplification process using pairs of flanked primers, a superprimer (10), and at least one first nicking enzyme. One pair of flanked primers is used for each target nucleic acid sequence to be amplified, and the pair of flanked primers is specific to the associated target nucleic acid sequence, each pair of flanked primers having a flanked forward primer and a flanked reverse primer; each flanked forward primer has a target-specific portion at the 3' end thereof and a a non-specific flank portion at the 5' end thereof, the target-specific portion being complementary to a portion of the associated target nucleic acid sequence; each flanked reverse primer has a target-specific portion at the 3' end thereof and a non-specific flank portion at the 5' end thereof, said target-specific portion being complementary to a portion of the associated target nucleic acid sequence; and the superprimer (10) has a binding portion (12) at the 3' end thereof and a recognition portion (14) at the 5' end thereof, said recognition portion (14) having a recognition sequence for the first nicking enzyme.

Description

[0001] Method, primer set and kit for the simultaneous amplification of multiple target nucleic acid sequences in a single sample

[0002] The invention relates to a method for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample. The invention further relates to a primer set and a kit for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample. The invention further relates to the use of the primer set and the use of the kit for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample.

[0003] The simultaneous amplification of multiple nucleic acids, such as DNA and RNA, in a single sample is of great importance in research and medicine. A key application is diagnostic testing, which allows for the simultaneous detection of multiple pathogens or clinically relevant biomarkers, such as tumor markers, in a single sample. Such multiplex testing saves time and money. The analysis is based on the detection of the amplified nucleic acids.

[0004] Methods for the simultaneous amplification of multiple nucleic acid sequences in a sample are known. These include multiplex PCR (polymerase chain reaction) methods, which use multiple primer pairs, each primer pair being specific for a particular nucleic acid sequence to be amplified. However, with an increasing number of primers, the formation of primer dimers and other undesirable interactions between the primers becomes more frequent, leading to reduced reaction efficiencies and limiting the number of nucleic acid sequences that can be amplified simultaneously.

[0005] WO 2005 / 038039 A2 discloses a multiplex PCR method for the simultaneous amplification of multiple nucleic acid sequences in a sample. In this method, known as "TemPCR," a pair of non-specific primers, called superprimers, is used in addition to multiple specific primer pairs. The superprimer pair consists of a forward superprimer and a reverse superprimer. The amplification reaction is primarily driven by the superprimers, thus reducing the concentration of the specific primers. This minimizes undesirable interactions between the primers in TemPCR. To precisely control the binding of the different primer types to the nucleic acid sequences during TemPCR, the specific primers and the superprimers are designed to have different melting temperatures.

[0006] Isothermal nucleic acid amplification methods, unlike PCR methods, take place at a constant temperature. They are particularly interesting for point-of-care analyses and patient-side diagnostic tests because, unlike PCR methods, they do not require temperature cycles and therefore do not require extensive equipment. Isothermal nucleic acid amplification methods are well-known. One example is strand displacement amplification, in which a DNA polymerase with strand displacement activity displaces an existing second strand of double-stranded DNA while synthesizing a new DNA strand using the first strand, which serves as a DNA template.

[0007] The use of isothermal amplification methods for the simultaneous amplification of multiple nucleic acid sequences in a sample is limited because, as the number of primers increases, primer dimers and other undesirable interactions between the primers also become more frequent, leading to reduced reaction efficiencies. Due to the constant temperature at which isothermal amplification methods are performed, the temperature profile during the amplification reaction cannot be adjusted, as is possible with TemPCR, to control the binding of different primer types to the nucleic acid sequences.

[0008] There is a need to provide further methods for the simultaneous amplification of multiple nucleic acid sequences in a sample.

[0009] The object of the invention is solved by a method for simultaneously amplifying a plurality of target nucleic acid sequences in a sample.

[0010] The method for simultaneously amplifying a plurality of target nucleic acid sequences in a sample comprises: (S300) amplifying the target nucleic acid sequences by isothermal strand displacement amplification using pairs of flanked primers, using a superprimer, and using at least one first nicking enzyme, wherein one pair of flanked primers is used for each target nucleic acid sequence to be amplified, and the pair of flanked primers is specific for the associated target nucleic acid sequence, wherein each pair of flanked primers has a flanked forward primer and a flanked reverse primer, wherein each flanked forward primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the associated target nucleic acid sequence.wherein each flanked backward primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, wherein the target-specific region is complementary to a region of the associated target nucleic acid sequence, wherein the superprimer has a binding region at its 3' end and a recognition region at its 5' end, wherein the recognition region has a recognition sequence for the first nicking enzyme, wherein for each pair of flanked primers individually one of the following variants is present:

[0011] (i) the flank section of the flanked forward primer is identical to the bonding section of the superprimer, and the flank section of the flanked reverse primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme,

[0012] (ii) the flank section of the flanked forward primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, and the flank section of the flanked reverse primer is identical to the binding section of the superprimer,

[0013] (iii) The flank region of the flanked forward primer is identical to the bonding region of the superprimer, and the flank region of the flanked reverse primer is identical to the bonding region of the superprimer. The method is a multiplex technique in which a plurality of target nucleic acid sequences are simultaneously amplified (multiplied) in a sample. The individual target nucleic acid sequences are all amplified together in the sample. In other words, the individual target nucleic acid sequences are amplified in a single reaction (and not in separate reactions (singleplex)). Compared to singleplex amplifications, the multiplex technique involves less effort and lower material consumption, thus saving time and costs.

[0014] The process is an in-vitro / intra-tro process.

[0015] The term "plural of target nucleic acid sequences" refers to two or more target nucleic acid sequences, particularly three or more. The method can be used, for example, to simultaneously amplify 2 to 10 target nucleic acid sequences, especially 3 to 10. More than 10 target nucleic acid sequences are also possible. Each target nucleic acid sequence typically has a length of 50 to 1,000 nucleotides.

[0016] The amplification of the target nucleic acid sequences is achieved via isothermal strand displacement amplification (SDA), also known as isothermal SDA. Amplification occurs at a constant reaction temperature (isothermal). Isothermal SDA is a well-known DNA amplification technique in which a DNA polymerase with strand displacement activity displaces an existing second strand of double-stranded DNA while synthesizing a new DNA strand using the first strand, which acts as a template. The synthesis of the new DNA strand occurs when the DNA polymerase continuously adds single nucleotides to a primer bound to the template. The new DNA strand then has the same nucleotide sequence as the second strand.Since these steps take place on both strands of the target nucleic acids, i.e., both strands of the target nucleic acids act as templates, an exponential amplification of the target nucleic acid sequences occurs.

[0017] Isothermal SDA is performed using multiple flanked primers as well as a superprimer. A flanked primer is an oligonucleotide that has a target-specific region and a non-specific flank region. The target-specific region is complementary to a segment of an associated target nucleic acid sequence. Thus, the target-specific region is specific with respect to an associated target nucleic acid sequence. The target-specific region can also be referred to as the sequence-specific region. The non-specific flank region, which can also be called the flank, is not complementary to a target nucleic acid sequence. The non-specific flank region can also be referred to as the non-target-specific region. Furthermore, the target-specific region can also be referred to as the complementary region and the non-specific flank region as the non-complementary region.The target-specific region is located at the 3' end of the flanked primer. The non-specific flank region is located at the 5' end of the flanked primer. The non-specific flank region serves to introduce an additional sequence segment into the amplification products. An intermediate segment, for example a spacer, can be arranged between the target-specific region and the non-specific flank region. Preferably, the flanked primers do not have an intermediate segment; that is, each flanked primer consists of the target-specific region and the non-specific flank region.

[0018] The target-specific section of the flanked primers has a length of at least 10 to approximately 30 nucleotides, preferably 15 to 25 nucleotides. For example, the target-specific section has a length of 20 nucleotides. The non-specific flank section of the flanked primers has a length of at least 10 to approximately 30 nucleotides, preferably 15 to 25 nucleotides. For example, the non-specific flank section has a length of 20 nucleotides. The flanked primers have a length of 20 to approximately 60 nucleotides.

[0019] Isothermal SDA is performed using pairs of flanked primers. One pair of flanked primers is used for each target nucleic acid sequence to be amplified. The pair of flanked primers is specific for the corresponding target nucleic acid sequence. Each pair of flanked primers has a flanked forward primer and a flanked reverse primer. Each flanked forward primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the corresponding target nucleic acid sequence. Each flanked reverse primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the corresponding target nucleic acid sequence.The target-specific region of the flanked forward primer binds to one of the two DNA strands of the corresponding target nucleic acid sequence, while the target-specific region of the flanked reverse primer binds to the other, complementary DNA strand of the corresponding target nucleic acid sequence. Thus, both strands of the corresponding target nucleic acid sequence are amplified in the isothermal SDA. There are various configurations for the non-specific flank region of the flanked forward primer and the non-specific flank region of the flanked reverse primer, which are described in more detail below. The pairs of flanked primers primarily serve to enrich the target nucleic acid sequences at the beginning of the SDA.

[0020] The superprimer is an oligonucleotide that, unlike flanked primers, does not have a target-specific region, meaning it does not have a region complementary to a region of a target nucleic acid sequence. The superprimer is completely non-specific with respect to target nucleic acid sequences. This applies to all target nucleic acid sequences to be amplified. The superprimer is universal. It is used for the amplification of all target nucleic acid sequences. Thus, it is possible to perform amplification with only one type of superprimer. The superprimer has a binding site and a recognition site. The binding site is located at the 3' end of the superprimer. The recognition site is located at the 5' end of the superprimer. An intermediate segment, such as a spacer, may be located between the binding site and the recognition site.Preferably, the superprimer has no intermediate section, i.e., the superprimer consists of the bonding section and the recognition section.

[0021] The bonding segment of the superprimer has a length of at least 10 to approximately 30 nucleotides, preferably 15 to 25 nucleotides. For example, the bonding segment has a length of 20 nucleotides. The recognition segment of the superprimer has a length of at least 10 to approximately 30 nucleotides, preferably 15 to 25 nucleotides. For example, the recognition segment has a length of 20 nucleotides. The superprimer has a length of 20 to approximately 60 nucleotides.

[0022] At the beginning of SDA, the superprimer cannot yet participate. Only when amplification products have been formed that possess a region complementary to the binding site of the superprimer, thus providing a binding site for the superprimer, can the superprimer participate in SDA. Such amplification products immediately function as templates for the subsequent amplification reaction via the superprimer. In the further course of SDA, the superprimer then primarily drives the exponential amplification of the target nucleic acid sequences.

[0023] Isothermal SDA occurs using at least one first nicking enzyme. A nicking enzyme (also called nickase) is an endonuclease that creates a single-strand break (nick) in double-stranded DNA. This process is also called "nicking." The single-strand break is created at a specific location within the double-stranded DNA, with the location being within or near a recognition sequence. The recognition sequence is specific to a particular nicking enzyme. The recognition sequence is typically 3 to 7 nucleotides long. The recognition sequence must be present in double-stranded DNA for the single-strand break to occur. The exact location of the single-strand break depends on the type of nicking enzyme.

[0024] The use of a nicking enzyme for isothermal SDA is well established. Once a double-stranded recognition sequence for the nicking enzyme has been generated during SDA, the nicking enzyme creates a single-strand break. At the site of the single-strand break (nicking site), DNA polymerase can bind again and synthesize the complementary strand, while the already existing complementary strand is displaced. The nicking enzyme and the arrangement of the double-stranded recognition sequence for the nicking enzyme generated during SDA are therefore selected such that the single-strand break is created in the strand that is to be extended by DNA polymerase starting from the single-strand break. Through repeated nicking and strand synthesis, exponential amplification of the target nucleic acid sequences occurs. For SDA, nicking enzymes are chosen that are active at the reaction temperature at which SDA is performed.Examples of suitable nicking enzymes are Nt.BspQI, Nt.Cvi-Pll, NtBstNBI, Nt.Alwl, Nt.BsmAI, Nt.BbvCI, Nb.BbvCI, Nb.Bsml, Nb.Bsml and Nb.BssSI, all from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA).

[0025] The recognition region of the superprimer contains a recognition sequence for the first nicking enzyme. This recognition sequence is shorter than the recognition region itself and therefore constitutes only a portion of it. The order of the recognition sequence within the recognition region is arbitrary. The recognition region of the superprimer does not contain a sequence identical to that of the binding region. Conversely, the binding region of the superprimer does not contain a recognition sequence for the first nicking enzyme.

[0026] Several variations are possible for the design of the non-specific flank section of the flanked forward primer and the non-specific flank section of the flanked reverse primer. For each pair of flanked primers, one of the following variations must be present:

[0027] (i) the flank section of the flanked forward primer is identical to the bonding section of the superprimer, and the flank section of the flanked reverse primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme,

[0028] (ii) the flank section of the flanked forward primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, and the flank section of the flanked reverse primer is identical to the binding section of the superprimer,

[0029] (iii) the flank section of the flanked forward primer is identical to the bonding section of the superprimer and the flank section of the flanked reverse primer is identical to the bonding section of the superprimer.

[0030] In variant (iii), the flank section of the flanked forward primer and the flank section of the flanked reverse primer are identical. This is not the case in variants (i) and (ii). In variants (i) and (ii), the flank section of one of the flanked primers in the pair of flanked primers is identical to the binding section of the superprimer, while the flank section of the other flanked primer in the pair of flanked primers contains the recognition sequence for the first nicking enzyme or the recognition sequence for a second nicking enzyme.

[0031] The flank section that is identical to the binding region of the superprimer does not contain a recognition sequence for the first nicking enzyme and, if a second nicking enzyme is used in step (S300), also does not contain a recognition sequence for the second nicking enzyme. The flank section that contains the recognition sequence for the first nicking enzyme or the recognition sequence for a second nicking enzyme does not contain a sequence identical to the binding region of the superprimer. The recognition sequence for the first nicking enzyme or for a second nicking enzyme is shorter than the flank section; therefore, it represents only a portion of the corresponding flank section. The arrangement of the recognition sequence within the corresponding flank section is arbitrary.It was observed that positioning the recognition sequence close to the target-specific region of the flanked primer is more effective than positioning it further away. This is likely because the DNA polymerase has to add fewer nucleotides the closer the recognition sequence is to the target-specific region of the flanked primer.

[0032] Each pair of flanked primers has at least one flank region that is identical to the binding site of the superprimer. All flank regions that are identical to the binding site of the superprimer are therefore also identical to each other. This allows the superprimer to bind equally to the amplification products of all target nucleic acid sequences. In this way, uniform amplification of all target nucleic acid sequences to be amplified by the superprimer can be ensured. More precisely, those strands of the target nucleic acid sequences whose amplification is driven by the superprimer are amplified to the same extent. Thus, only a single primer, namely the superprimer, is required for the exponential amplification of all target nucleic acid sequences.Furthermore, this approach overcomes any existing differences in amplification efficiencies between the individual target nucleic acid sequences, which would be detrimental to a multiplex amplification process. In variant (iii), recognition by the first nicking enzyme occurs exclusively via the recognition sequence introduced into the amplification products by the superprimer. Therefore, at the beginning of SDA, amplification products without a recognition sequence for a nicking enzyme are initially formed. Only after a second reaction step has synthesized a segment complementary to the flank region of the flanked forward primer and a segment complementary to the flank region of the flanked reverse primer, can the superprimer bind and thus incorporate the recognition segment of the superprimer into the amplification products of a third reaction step.In the further course of the SDA, the recognition sections of the superprimer are present in a double-stranded configuration. Single-strand breaks can then be generated on these resulting double-stranded recognition sequences.

[0033] In variant (i), the extension of the flanked forward primer in a first reaction step initially produces an amplification product without a recognition sequence for a nicking enzyme. Only after a complementary segment to the flank of the flanked forward primer is synthesized in a second reaction step can the superprimer bind, thus incorporating the superprimer's recognition segment into the amplification product of a third reaction step. In contrast, the flank of the flanked reverse primer contains the recognition sequence for the first nicking enzyme or for a second nicking enzyme, so that the corresponding recognition sequence is already present in a double strand after the second reaction step, leading to a single-strand break. In the subsequent course of the SDA, both the recognition segment of the superprimer and the flank of the flanked reverse primer are then present in a double strand.Single-strand breaks can now be generated in both of the resulting double-stranded recognition sequences.

[0034] In variant (ii), the extension of the flanked reverse primer in a first reaction step initially produces an amplification product without a recognition sequence for a nicking enzyme. Only after a complementary segment to the flank of the flanked reverse primer is synthesized in a second reaction step can the superprimer bind, thus incorporating the superprimer's recognition segment into the amplification product of a third reaction step. The flank of the flanked forward primer, on the other hand, contains the recognition sequence for the first nicking enzyme or for a second nicking enzyme, so that the corresponding recognition sequence is already present in a double strand after the second reaction step, leading to a single-strand break. In the subsequent course of the SDA, both the recognition segment of the superprimer and the flank of the flanked forward primer are then present in a double strand.Single-strand breaks can now be generated in both of the resulting double-stranded recognition sequences.

[0035] In all three variants (i), (ii), and (iii), the recognition sequence for the first nicking enzyme required for the efficient progression of the amplification reaction is introduced into the SDA via the superprimer for one of the two strands (variants (i) and (ii)) or for both strands (variant (iii)) of each target nucleic acid sequence. This means that the first nicking enzyme can only generate the single-strand break once the superprimer has been incorporated into the amplification products of the target nucleic acid sequences and the sequence region containing the recognition sequence for the first nicking enzyme is present in a double-stranded form. Following the single-strand break, another amplification step takes place.

[0036] In variant (i) and variant (ii), one of the two strands of the target nucleic acid sequence is preferentially amplified using the superprimer, while in variant (iii) both strands of the target nucleic acid sequence are amplified to the same extent using the superprimer.

[0037] In variant (i), the binding site of the superprimer allows the superprimer to bind to a newly synthesized DNA strand that has a region complementary to the flanked forward primer. As a result, in variant (i), the superprimer promotes the amplification of the strand to which the flanked forward primer binds (at least initially during SDA). This leads to the preferential amplification of this strand in variant (i).

[0038] In variant (ii), the binding site of the superprimer allows it to bind to a newly synthesized DNA strand that has a region complementary to the flanked reverse primer. In variant (ii), the superprimer thus promotes the amplification of the strand to which the flanked reverse primer binds (at least initially during SDA). This results in this strand being preferentially amplified in variant (ii). Variants (i) and (ii) may be of interest, for example, if the detection of the amplification products obtained in step (S300) is based on only one of the two strands of the target nucleic acid sequence associated with the corresponding pair of flanked primers. Depending on which strand the detection is based on, choosing variant (i) or variant (ii) can achieve preferential amplification of that strand.A typical example is detection using a labeled probe, such as a fluorescently labeled probe, which binds to only one of the two strands of the target nucleic acid sequence. Thus, only one of the two strands of the target nucleic acid sequence is detected by the labeled probe. Therefore, only one of the two strands of the target nucleic acid sequence is required for a positive detection signal. By choosing variant (i) or variant (ii), the reaction equilibrium of the SDA can be adjusted such that the probe-binding strand of the target nucleic acid sequence is preferentially amplified. This results in a stronger detection signal compared to amplification of both strands to the same extent for the same amplification time. This is particularly useful for real-time detection of the amplification products obtained in step (S300).Furthermore, smaller amounts of reagents are sufficient if the strand of the target nucleic acid sequence required for a positive detection signal is preferentially amplified.

[0039] In variant (iii), the binding site of the superprimer allows the superprimer to bind to both a newly synthesized DNA strand that has a region complementary to the flanked forward primer and a newly synthesized DNA strand that has a region complementary to the flanked reverse primer. Thus, in variant (iii), the superprimer promotes amplification of both the strand to which the flanked forward primer binds (at least initially during SDA) and the strand to which the flanked reverse primer binds (at least initially during SDA). Variant (iii) may be of interest, for example, if the amplification products obtained in step (S300) are to be used further.

[0040] In variant (iii), only one type of nicking enzyme is used, namely the first nicking enzyme. Variants (i) and (ii) also allow the use of only one type of nicking enzyme, namely the first nicking enzyme. Alternatively, variants (i) and (ii) can use two different types of nicking enzymes: the first nicking enzyme and the second nicking enzyme. Different types of nicking enzymes have different recognition sequences. When using different types of nicking enzymes, the recognition sequence for the first nicking enzyme is located on the recognition site of the superprimer, and the recognition sequence for the second nicking enzyme is located on the flanking site of the flanked reverse primer (variant (i)) or on the flanking site of the flanked forward primer (variant (ii)).The first and second Nicking enzymes can be chosen, for example, to exhibit different activities at the reaction temperature at which SDA is performed. For instance, the first Nicking enzyme might have a higher activity than the second at this reaction temperature. In other words, the temperature optimum of the first Nicking enzyme is closer to the reaction temperature at which SDA is performed than the temperature optimum of the second Nicking enzyme. This results in the preferentially amplified strand being amplified even more strongly. This can be of interest, for example, if the detection of the amplification products obtained in step (S300) is based on only one of the two strands of the target nucleic acid sequence associated with the corresponding pair of flanked primers.

[0041] Since the selection of one of the variants from (i), (ii) and (iii) is made individually for each pair of flanked primers, it is also possible that the type of second nicking enzyme differs between different pairs of flanked primers.

[0042] If only one type of nicking enzyme, namely the first nicking enzyme, is used, the recognition region of the superprimer and the flank region containing the recognition sequence for the first nicking enzyme (i.e., the flank region of the flanked reverse primer in variant (i) or the flank region of the flanked forward primer in variant (ii)) can be identical or different except for the recognition sequence for the first nicking enzyme. If these regions differ except for the recognition sequence for the first nicking enzyme, the accumulation of identical sequence segments across multiple primers is reduced. This lowers the risk of unwanted interactions between the primers. The position of the recognition sequence within these regions can also differ. This applies accordingly to the flank regions containing the recognition sequence for the same nicking enzyme.This means that if several pairs of flanked primers are present in variant (i) and / or variant (ii), and the same nicking enzyme is used for these pairs, then the corresponding flank regions containing the recognition sequence for the nicking enzyme can be identical or different except for the recognition sequence. If these regions differ except for the recognition sequence, the accumulation of identical sequence regions across multiple primers is reduced. This lowers the risk of unwanted interactions between the primers. The position of the recognition sequence within these regions can also differ.

[0043] As already described, the position of the single-strand break generated by the nicking enzyme, i.e., the nicking site, depends on the type of nicking enzyme. It lies within or near the recognition sequence for the nicking enzyme. Thus, the following possibilities exist: (1) the position of the single-strand break lies within the recognition sequence, (2) the position of the single-strand break lies to the left of the recognition sequence, or (3) the position of the single-strand break lies to the right of the recognition sequence. In possibility (1), the position of one single-strand break lies in the recognition region of the superprimer, and, in the case of variants (i) and (ii), the position of another single-strand break lies in the flank region of the flanked primer that contains the recognition sequence for the nicking enzyme (i.e., in the flank region of the flanked backward primer in variant (i) or in the flank region of the flanked forward primer in variant (ii)).Depending on the position of the recognition sequence for the nicking enzyme within the aforementioned sections, the position of the single-strand break may also lie within the aforementioned sections in possibilities (2) and (3) (but, unlike possibility (1), outside the recognition sequence). Furthermore, in possibilities (2) and (3), the position of the single-strand break may also lie outside the aforementioned sections, for example, in the target-specific section of the flanked primer whose flank section contains the recognition sequence for the nicking enzyme. For the execution of SDA, the precise location of the single-strand break is generally irrelevant.

[0044] For SDA, the target nucleic acid sequences are combined with the pairs of flanked primers, the superprimer, the first nicking enzyme, and, if applicable, the second (and any further) nicking enzyme(s). Furthermore, the target nucleic acid sequences are combined with a DNA polymerase with strand displacement activity in a suitable buffer compatible with the DNA polymerase, as well as with nucleotides (deoxynucleotide triphosphates (dNTPs)). DNA polymerases with strand displacement activity are known. Examples include the SD polymerase from Bioron GmbH (Bioron GmbH, Römerberg, Germany), the Bst 2.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), the Bst 3.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), the BST +DNA polymerase from ArcticZymes Technologies ASA (ArcticZymes Technologies ASA, Tromso, Norway), and the phi29 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA).

[0045] For SDA, reaction conditions, particularly a reaction temperature, are selected that promote both the binding of the target-specific regions of the flanked primers to the target nucleic acid sequences and the binding of the superprimer's binding site to complementary regions on amplification products. The binding of the target-specific regions of the flanked primers to the target nucleic acid sequences is especially important at the beginning of SDA because the superprimer cannot yet participate in the process. As SDA progresses, the binding of the superprimer to complementary regions on amplification products becomes more significant.

[0046] The reaction temperature at which SDA is performed is typically chosen to best support the binding of the superprimer's bonding region to complementary regions on amplification products. The reaction temperature can range from approximately 30°C to approximately 70°C. Preferably, the reaction temperature is in the range of 37°C to 65°C. For example, the reaction temperature is 55°C.

[0047] SDA begins by binding the target-specific segments of the flanked primers to the corresponding target nucleic acid sequences. The DNA polymerase with strand displacement activity continuously adds single nucleotides 5' to 3' towards the flanked primers, creating a new strand in each case. At the end of this first reaction step, two newly synthesized DNA strands are present for each target nucleic acid sequence, each exhibiting the flank segment of the corresponding flanked primer at its 5' end. In a second reaction step, these newly synthesized strands serve as a template to which the opposite flanked primer of the corresponding pair of flanked primers binds. In variant (i), if the flanked forward primer is extended in the first reaction step, an amplification product without a recognition sequence for a nicking enzyme is initially formed.The newly synthesized DNA strand from the second reaction step has a segment complementary to the flanked forward primer. This segment complementary to the flanked forward primer includes the segment complementary to the flank of the flanked forward primer, to which the binding site of the superprimer binds in a third reaction step. The superprimer is elongated 5' to 3'-wise by DNA polymerase, resulting in a newly synthesized DNA strand that has the recognition region of the superprimer at its 5' end. A fourth reaction step follows, after which the recognition region of the superprimer is now double-stranded, leading to a single-strand break caused by the first nicking enzyme. At the site of the single-strand break, DNA polymerase can bind again and synthesize the complementary strand, while the already present complementary strand is displaced.In contrast to the extension of the flanked forward primer, in the case of the extension of the flanked reverse primer in variant (i), an amplification product is formed in the first reaction step that already contains the recognition sequence for the first nicking enzyme or for a second nicking enzyme. After the second reaction step, this recognition sequence is present in a double strand, so that a single-strand break occurs immediately after the second reaction step due to the corresponding nicking enzyme. At the site of the single-strand break, the DNA polymerase can bind again and synthesize the complementary strand, while the already present complementary strand is displaced. Through repeated nicking and strand synthesis, an exponential amplification of the target nucleic acid sequences takes place.

[0048] In variant (ii), the described sequence of the first reaction steps of the SDA applies accordingly, whereby the roles of flanked forward primer and flanked backward primer are reversed compared to variant (i).

[0049] In variant (iii), the described sequence of the first reaction steps of the SDA also applies accordingly, whereby the sequence described for the flanked forward primer in variant (i) also applies to the flanked backward primer in variant (iii).

[0050] The method according to the invention enables the simultaneous amplification of a plurality of target nucleic acid sequences in a sample, with efficient amplification of the plurality of target nucleic acid sequences taking place. Due to the plurality of target nucleic acid sequences, a large number of specific primers are required. Without the use of a superprimer, these specific primers would have to be used at a concentration that would lead to increased formation of primer dimers and other undesirable interactions between the primers. Such interactions would result in reduced reaction efficiencies, so that efficient amplification of the plurality of target nucleic acid sequences would not be possible.While the use of the superprimer increases the number of different primers in the amplification reaction by one, the concentrations of the specific primers (i.e., the flanked primers and, if applicable, external primers, which will be described in more detail below) can be reduced so significantly that the undesired interactions between the primers are considerably diminished or even eliminated. Thus, the method enables the efficient amplification of the majority of target nucleic acid sequences.

[0051] Compared to a process without a superprimer, the concentration of the specific primer used in the highest concentration among the other specific primers can be reduced to approximately 1 / 5 to 1 / 10. This applies accordingly if all specific primers are used in the same concentration or if there are several specific primers used in the same highest concentration among the other specific primers. If applicable, the concentrations of the other specific primers that are not used in the highest concentration among the other specific primers are also reduced accordingly.

[0052] The reduction in the concentration of specific primers (that is, flanked primers and, if applicable, external primers, which will be described in more detail below) is possible because the specific primers are only needed at the beginning of the amplification reaction to enrich the target nucleic acid sequences and to incorporate the non-specific flank region, which is identical to the binding site of the superprimer, into at least some of the amplification products. The binding site of the superprimer can then bind to its complementary region during the subsequent SDA reaction and drive the exponential amplification of the target nucleic acid sequences.

[0053] At the same time, the method according to the invention enables isothermal amplification of the majority of target nucleic acid sequences. Therefore, no temperature cycles are required, and consequently, no significant equipment complexity is necessary. The different primer types can all bind to the corresponding complementary nucleic acid sequences at the reaction temperature at which the amplification is carried out.

[0054] This method requires only a single type of superprimer for the exponential amplification of all target nucleic acid sequences. Therefore, it enables the exponential amplification of all target nucleic acid sequences using only one type of superprimer. This eliminates the need for two types of superprimers, such as a forward and a reverse superprimer as in TemPCR. In this way, the total number of required primers is kept to a minimum.

[0055] The superprimer contains the recognition sequence for the first nicking enzyme required for SDA. As previously described, the first nicking enzyme can only generate the single-strand break once the superprimer has been incorporated into the amplification products of the target nucleic acid sequences and the sequence region containing the recognition sequence for the first nicking enzyme is present in a double-stranded form. Following the single-strand break, a further amplification step takes place.

[0056] As previously described, this method enables efficient amplification of the majority of target nucleic acid sequences. This means that SDA can be performed for a duration of, for example, approximately 20 minutes. In the case of real-time detection of the amplification products obtained in the SDA, positive detection signals could be obtained after only 5 to 15 minutes. To reliably determine the absence of a specific target nucleic acid sequence, for example, in a diagnostic test, SDA can be performed for a duration of 20 to 25 minutes. Such a short procedure duration is particularly important for diagnostic tests. Depending on the application, the duration of SDA can also be significantly longer, for example, from 1.5 to 18 hours.

[0057] According to another embodiment, each pair of flanked primers has either variant (i) or variant (ii). In this way, only one flanked primer per pair of flanked primers has a flank region that is identical to the binding region of the superprimer. If the flank region of the other flanked primer per pair of flanked primers—that is, the flank region containing the recognition sequence for the first nicking enzyme or the recognition sequence for a second nicking enzyme—is chosen so that it is not identical to the recognition region of the superprimer, a smaller number of identical sequence regions are introduced into the SDA than if variant (iii) were included. For each target nucleic acid sequence, one of the two strands of the target nucleic acid sequence is preferably amplified using the superprimer.This may be of interest, for example, if the detection of the amplification products obtained in step (S300) is based on only one of the two strands of the target nucleic acid sequences.

[0058] According to another embodiment, variant (i) is present for all pairs of flanked primers. In this way, all flanked forward primers have the flanking region that is identical to the binding region of the superprimer. This simplifies the design of the flanked primers. For each target nucleic acid sequence, the strand of the target nucleic acid sequence is preferably amplified by the superprimer, to which the flanked forward primer binds (at least at the beginning of the SDA).

[0059] According to another embodiment, variant (ii) is present for all pairs of flanked primers. In this way, all flanked reverse primers have a flank segment that is identical to the binding segment of the superprimer. This simplifies the design of the flanked primers. For each target nucleic acid sequence, the strand of the target nucleic acid sequence is preferably amplified by the superprimer, to which the flanked reverse primer binds (at least at the beginning of the SDA).

[0060] According to another embodiment, variant (iii) is present for all pairs of flanked primers. In this embodiment, all flanked primers have a flank region that is identical to the binding region of the superprimer. This simplifies the design of the flanked primers. Both strands of the target nucleic acid sequences are amplified equally by the superprimer.

[0061] According to a further embodiment, step (S300) additionally includes the detection of the amplification products obtained in this step. This enables real-time detection of the amplification products. The amplification products can, for example, be detected optically in real time. Step (S300) can be performed, for example, using detection probes, with one detection probe being used for each target nucleic acid sequence to be amplified and the detection probe being specific for the corresponding target nucleic acid sequence. A detection probe, which can also be referred to as a hybridization probe or detection probe, is an oligonucleotide that is complementary to a segment of a target nucleic acid sequence to be amplified and that has a label which, upon binding of the oligonucleotide to this segment, generates a detectable signal, in particular an optically detectable signal.When using multiple detection probes, such as one probe per target nucleic acid sequence to be amplified, each probe generates a signal specific to it. The optically detectable signal can, for example, be a fluorescence signal. Fluorescence-labeled detection probes suitable for isothermal amplification methods are known.

[0062] The detection probes can, for example, be "molecular beacons." A molecular beacon consists of a single-stranded DNA molecule with a stem-loop structure. A fluorescent dye and a quencher are located at the stem-related 3' and 5' ends, respectively. The loop contains a DNA sequence complementary to the amplification product to be detected. In its non-hybridized state, the molecular beacon exists in the stem-loop structure. In this state, the fluorescence of the fluorescent dye is suppressed by energy transfer to the quencher based on Förster resonance energy transfer. Hybridization of the molecular beacon with the amplification product to be detected causes the stem-loop structure to break down, thus reducing the energy transfer from the fluorescent dye to the quencher.In this way, hybridization leads to an increase in fluorescence, which can be used for real-time detection of the amplification product.

[0063] According to an alternative embodiment, the method further comprises: (S500) detection of the amplification products obtained in step (S300).

[0064] In this case, the amplification products obtained in step (S300) are detected by a downstream detection method. Such detection methods are known. These include, for example, detection of the amplification products by DNA gel electrophoresis (agarose gel electrophoresis) or detection of the amplification products by hybridization on microarrays.

[0065] According to another embodiment, the method further comprises:

[0066] (S200) Heating the sample to separate double-stranded target nucleic acid sequences into their single strands.

[0067] Step (S200) involves the denaturation of double-stranded target nucleic acid sequences. The sample is typically heated at 95°C for approximately 1 to 3 minutes. Afterward, the sample is cooled or allowed to cool to the reaction temperature at which the SDA is performed. Step (S200) promotes the binding of the flanked primers to the complementary regions of the corresponding target nucleic acid sequences, thus facilitating the initiation of the SDA reaction.

[0068] The separation of double-stranded target nucleic acid sequences into their single strands can alternatively be carried out using another known method, for example by enzymatic denaturation using helicases or by chemical denaturation.

[0069] According to another embodiment, the method further comprises:

[0070] (S100) Reverse transcription of target nucleic acid sequences in the form of RNA into complementary DNA (cDNA).

[0071] Step (S100) is required if at least one of the target nucleic acid sequences is present in the form of RNA, for example, mRNA. Reverse transcription is a well-known molecular biology technique. Step (S100) can be performed together with SDA as a one-step assay in the same reaction mixture. Alternatively, reverse transcription can be performed in a separate step preceding the procedure (two-step assay). According to another embodiment, the procedure further comprises:

[0072] (S400) Cooling of the amplification products obtained in step (S300).

[0073] Step (S400) allows the amplification products obtained in step (S300) to be used or stored for a downstream detection method or for other purposes. Typically, the reaction mixture is cooled to 4°C. If the sole purpose is the detection of the amplification products obtained in step (S300), and this is performed, for example, by real-time detection, step (S400) is not required.

[0074] According to another embodiment, the reaction mixture from step (S300) is briefly heated again before step (S400), for example for about 1 minute, to inactivate the DNA polymerase and the nicking enzyme. This can protect the amplification products obtained in step (S300) from degradation by the nicking enzyme.

[0075] According to a further embodiment, step (S300) is performed using pairs of external primers, wherein one pair of external primers is used for each target nucleic acid sequence to be amplified, and the pair of external primers is specific for the associated target nucleic acid sequence, wherein each pair of external primers comprises an external forward primer and an external reverse primer, wherein the external forward primer is complementary to a section of the associated target nucleic acid sequence that lies 3'-ward to the section to which the sequence-specific section of the flanked forward primer associated with that target nucleic acid sequence is complementary, and wherein the external reverse primer is complementary to a section of the associated target nucleic acid sequence that lies 3'-ward to the sectionto which the sequence-specific section of the flanked reverse primer belonging to this target nucleic acid sequence is complementary.

[0076] An external primer is a target-specific oligonucleotide that is complementary to a segment of a corresponding target nucleic acid sequence. The external primer is therefore specific with respect to its corresponding target nucleic acid sequence. Unlike flanked primers, external primers do not have a non-specific flank segment. External primers can also be called outer primers because, compared to their corresponding flanked primers, they bind "further out" to the corresponding target nucleic acid sequences. External primers typically have a length of at least 10 to approximately 40 nucleotides, preferably 15 to 25 nucleotides. For example, an external primer has a length of 20 nucleotides.

[0077] The use of external primer pairs serves to facilitate the binding of the flanked primers to the complementary regions of the corresponding target nucleic acid sequences within the DNA double helix, thereby accelerating the initiation of the SDA reaction. The use of external primer pairs leads to a significant improvement in the first amplification reactions. However, the use of external primer pairs is not mandatory. The inventive method can also be performed without external primers.

[0078] According to another embodiment, the superprimer is used at a higher concentration than the flanked primers. For example, the superprimer can be used at a concentration approximately 10 to 100 times higher than the concentration(s) of the flanked primers. The superprimer is thus used in excess. Using the superprimer at a higher concentration compared to the concentration(s) of the flanked primers promotes the progression of SDA, since SDA is primarily driven by the superprimer. If additional pairs of external primers are used, then the superprimer is used at a higher concentration than both the flanked primers and the external primers. In other words, the superprimer is then used at a higher concentration than any other primer in the SDA.

[0079] For example, the concentrations of the superprimer and the flanked primers are chosen such that the superprimer is present at a concentration suitable for promoting exponential amplification of the target nucleic acid sequences, while the flanked primers are present at a concentration insufficient for this purpose. For example, the superprimer can be used at a concentration of 0.5 to 2.5 pM. The flanked primers can be used at a concentration of 0.02 to 0.15 pM.

[0080] According to another embodiment, the superprimer is used at a concentration approximately 15 to 25 times higher than the concentration of one of the flanked primers used at the highest concentration among the other flanked primers. This applies accordingly if there are several flanked primers used at the same highest concentration among the other flanked primers. As will be explained in more detail below, the flanked primers can be used at different concentrations. Based on the highest concentration used in each of these cases, the concentration of the superprimer in this embodiment is approximately 15 to 25 times higher. In other words, the concentration of the superprimer is approximately 15 to 25 times the highest concentration used among the other flanked primers. The superprimer is thus used in a large excess to promote the progression of the SDA (Single-Diffusion Application).

[0081] The flanked primers can be used together at the same concentration or at different concentrations. This applies both across pairs of flanked primers and within a pair of flanked primers. For example, in variant (i), the flanked forward primer of a pair of flanked primers can be used at a higher concentration than the flanked reverse primer of the pair of flanked primers, and in variant (ii), the flanked reverse primer of a pair of flanked primers can be used at a higher concentration than the flanked forward primer of the pair of flanked primers.

[0082] According to another embodiment, in variant (i) the flanked reverse primer is used in a concentration that is about 0.5 to 0.75 times higher than the concentration of the flanked forward primer.

[0083] According to another embodiment, in variant (ii) the flanked forward primer is used in a concentration that is about 0.5 to 0.75 times higher than the concentration of the flanked reverse primer.

[0084] In both cases, the differing concentrations of the flanked primers of a pair of flanked primers favor the preferential amplification of one of the two strands of the target nucleic acid sequence associated with the corresponding pair of flanked primers, even at the beginning of SDA. For example, in detection using a labeled probe, the probe-binding strand can be selected as the preferentially amplified strand. This is particularly useful for real-time detection of the amplification products obtained in step (S300). As described above, in variant (i), the preferentially amplified strand of the target nucleic acid sequence is the strand to which the flanked forward primer binds (at least at the beginning of SDA), and in variant (ii), it is the strand to which the flanked reverse primer binds (at least at the beginning of SDA).

[0085] In variant (iii) the flanked forward primer of a pair of flanked primers can also be used in a different concentration than the flanked reverse primer of the pair of flanked primers.

[0086] If additional pairs of external primers are used, they are typically used at a lower concentration than the flanked primers. This is because the external primers only play a role in initiating the SDA reaction. The external primers can be used at the same concentration or at different concentrations, both across and within pairs of external primers. Typically, external primers are used at the same concentration.

[0087] According to a further embodiment, in at least one, but not all, pairs of flanked primers, an additional segment is arranged at the 5' end of the flank region, which is identical to the bonding region of the superprimer. The sequence of the additional segment is identical to the portion of the recognition region of the superprimer following the bonding region, corresponding to the length of the additional segment. The additional segment comprises at least one nucleotide. The additional segment causes the melting temperature of the superprimer to be selectively increased for the corresponding target nucleic acid sequence(s). In this way, the binding of the superprimer to the other target nucleic acid sequence(s) or to their amplicons is favored. The reaction equilibrium is thus shifted in favor of these target nucleic acid sequence(s).

[0088] The superprimer preferentially binds to the target nucleic acid sequence(s) or their amplicons, provided that no additional segment has been incorporated due to the structure of their corresponding flanked primers. This is because the reaction temperature at which superprimer dissociation (SDA) is performed is typically chosen to optimally support the binding of the superprimer's binding site to complementary sites on the amplification products. If the binding site of the superprimer becomes elongated due to the use of the additional segment, then the reaction temperature at which SDA is performed is no longer optimal for this binding, making it more difficult for the superprimer to bind completely. Therefore, the use of the additional segment is only suitable for those target nucleic acid sequences that are intended to be amplified less strongly than the majority of target nucleic acid sequences.Such a target nucleic acid sequence can, for example, be a target nucleic acid sequence to be amplified, which merely serves as a control. Using a control, it can be determined that SDA has actually taken place (positive control). The amplification of the target nucleic acid sequence(s) not serving as a control then occurs preferentially compared to the control.

[0089] The invention further relates to a primer set for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample, the primer set comprising:

[0090] (a) pairs of flanked primers, and

[0091] (b) a superprimer, wherein one pair of flanked primers is provided for each target nucleic acid sequence to be amplified, and the pair of flanked primers is specific for the corresponding target nucleic acid sequence, each pair of flanked primers comprising a flanked forward primer and a flanked reverse primer, each flanked forward primer having a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the corresponding target nucleic acid sequence, each flanked reverse primer having a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the corresponding target nucleic acid sequence,wherein the superprimer has a binding section at its 3' end and a recognition section at its 5' end, the recognition section having a recognition sequence for a first nicking enzyme, wherein for each pair of flanked primers individually one of the following variants is present:,

[0092] (i) the flank section of the flanked forward primer is identical to the binding section of the superprimer, and the flank section of the flanked reverse primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme,

[0093] (ii) the flank section of the flanked forward primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, and the flank section of the flanked reverse primer is identical to the binding section of the superprimer,

[0094] (iii) the flank section of the flanked forward primer is identical to the bonding section of the superprimer and the flank section of the flanked reverse primer is identical to the bonding section of the superprimer.

[0095] According to another embodiment, the primer set further comprises:

[0096] (c) Pairs of external primers, wherein there is one pair of external primers for each target nucleic acid sequence to be amplified and the pair of external primers is specific for the associated target nucleic acid sequence, wherein each pair of external primers comprises an external forward primer and an external reverse primer, wherein the external forward primer is complementary to a section of the associated target nucleic acid sequence that lies 3'-ward to the section to which the sequence-specific section of the flanked forward primer associated with that target nucleic acid sequence is complementary, and wherein the external reverse primer is complementary to a section of the associated target nucleic acid sequence that lies 3'-ward to the section to which the sequence-specific section of the flanked reverse primer associated with that target nucleic acid sequence is complementary.

[0097] The further explanations and embodiments disclosed in the description of the method, in particular with regard to the pairs of flanked primers, the superprimer and the external primers, apply accordingly to the primer set.

[0098] The invention further relates to the use of the primer set according to the invention for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample. The further explanations and embodiments disclosed in the description of the method apply accordingly to the use of the primer set.

[0099] The invention further relates to a kit for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample, the kit comprising:

[0100] (a) Primer set according to the invention, and

[0101] (b) the first nicking enzyme.

[0102] The kit may also include a second (and possibly further) nicking enzyme(s), especially if the primer set includes a flanked primer with a corresponding recognition sequence.

[0103] According to another embodiment, the kit further includes:

[0104] (c) a DNA polymerase with strand displacement activity,

[0105] (d) a buffer compatible with DNA polymerase, and

[0106] (e) Deoxynucleotide triphosphates.

[0107] The kit can also include a detection system for detecting the amplification products, i.e., the amplified target nucleic acid sequences. This detection system can, for example, be based on detection probes.

[0108] According to another embodiment, the kit further includes:

[0109] (f) Detection probes, wherein there is one detection probe for each target nucleic acid sequence to be amplified and the detection probe is specific for the corresponding target nucleic acid sequence.

[0110] The further explanations and embodiments disclosed in the description of the method, in particular with regard to the individual components of the kit, apply accordingly to the kit.

[0111] The invention further relates to a use of the kit according to the invention for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample.

[0112] The further explanations and embodiments disclosed in the description of the method apply accordingly to the use of the kit. The invention will now be explained with reference to the figures. They show:

[0113] Figure 1 schematically represents a process flow of a method for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample.

[0114] Figure 2 shows a schematic representation of an embodiment of a superprimer.

[0115] Figure 3 in schematic representation embodiments of a pair of flanked primers, for which variant (i) is present (Figure 3A), a pair of flanked primers, for which variant (ii) is present (Figure 3B), and a pair of flanked primers, for which variant (iii) is present (Figure 3C).

[0116] Figure 4 schematically represents a part of the process sequence shown in Figure 1, where the part relates to the beginning of the isothermal strand displacement amplification.

[0117] With reference to Figures 1 to 4, a procedure for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample is explained.

[0118] The method is a multiplex method in which the target nucleic acid sequences are amplified simultaneously in one sample, i.e., in the same reaction mixture.

[0119] In the present embodiment, the number of target nucleic acid sequences that are amplified simultaneously is three. However, the method can also be used for only two target nucleic acid sequences or for more than three target nucleic acid sequences.

[0120] In a first step (S200), the sample containing the three target nucleic acid sequences is heated to 95°C for 1 minute. This causes denaturation, meaning that the target nucleic acid sequences, which are present as double-stranded DNA, are separated into their single strands. Denaturation promotes the binding of primers to complementary regions of the target nucleic acid sequences and thus the initiation of the isothermal strand displacement amplification (SDA) reaction, which follows in a subsequent step (S300). After heating, the sample is cooled to the reaction temperature at which the SDA is performed. In this embodiment, the reaction temperature is 55°C. In contrast to this embodiment, step S200 can also be omitted, for example, if the sample is already provided in denatured form.

[0121] In step S300, the target nucleic acid sequences are simultaneously amplified using isothermal SDA. The amplification takes place at a constant temperature of 55°C. SDA is performed using pairs of flanked primers. One pair of flanked primers is used for each target nucleic acid sequence to be amplified. The pair of flanked primers is specific to the corresponding target nucleic acid sequence. In the present embodiment, three pairs of flanked primers are used: a first pair specific for one of the three target nucleic acid sequences, a second pair specific for a second target nucleic acid sequence, and a third pair specific for a third target nucleic acid sequence.Each pair of flanked primers comprises a flanked forward primer and a flanked reverse primer. In total, six flanked primers are used in this embodiment.

[0122] Each flanked primer has two sections: a target-specific section located at its 3' end and a non-specific flank section located at its 5' end. The target-specific section is complementary to a segment of the target nucleic acid sequence for which the flanked primer is specific. This is not the case for the flank section. Thus, when the flanked primer binds to the corresponding target nucleic acid sequence, only the target-specific section of the flanked primer binds to the target nucleic acid sequence (see Figure 4 with first flanked forward primer 20 and first flanked reverse primer 30). In contrast to the present embodiment, an intermediate section can be arranged between the target-specific section and the non-specific flank section.

[0123] SDA is performed using three pairs of external primers (one pair per target nucleic acid sequence to be amplified): a first pair specific for the first target nucleic acid sequence, a second pair specific for the second target nucleic acid sequence, and a third pair specific for the third target nucleic acid sequence. Each pair of external primers includes one external forward primer and one external reverse primer. In total, six external primers are used in this embodiment. Alternatively, the pairs of external primers can be omitted.

[0124] Each external primer is complementary to a segment of the target nucleic acid sequence for which the external primer is specific. Upon binding of the external primer to the corresponding target nucleic acid sequence, the entire external primer binds to the target nucleic acid sequence (see Figure 4 with first external forward primer 40 and first external reverse primer 50). The external forward primer is complementary to a segment of the corresponding target nucleic acid sequence that lies 3'-side to the segment to which the target-specific segment of the flanked forward primer associated with that target nucleic acid sequence is complementary.The external reverse primer is complementary to a section of the associated target nucleic acid sequence that lies 3'-ward to the section to which the target-specific section of the flanked reverse primer associated with that target nucleic acid sequence is complementary (see Figure 4 with first external forward primer 40, first flanked forward primer 20, first external reverse primer 50 and first flanked reverse primer 30).

[0125] SDA is also performed using a superprimer 10. Figure 2 shows a schematic representation of the structure of the superprimer 10. The superprimer has two sections 12 and 14: a binding section 12, located at the 3' end of the superprimer 10, and a recognition section 14, located at the 5' end of the superprimer 10. The recognition section 14 has a recognition sequence for a first nicking enzyme. In contrast to the present embodiment, an intermediate section can be arranged between the binding section 12 and the recognition section 14.

[0126] Each pair of flanked primers exhibits one of the variants (i), (ii), or (iii). Figure 3 schematically illustrates the corresponding structures of the pairs of flanked primers. Figure 3A shows the structure of a pair of flanked primers 20, 30, for which variant (i) is present. This is, by way of example, the first pair of flanked primers specific for the first target nucleic acid sequence. The pair comprises a first flanked forward primer 20 and a first flanked reverse primer 30. The first flanked forward primer 20 has two sections 22, 24: a target-specific section 22 located at the 3' end of the first flanked forward primer 20, and a non-specific flank section 24 located at the 5' end of the first flanked forward primer 20.The first flanked reverse primer 30 has two sections 32 and 34: a target-specific section 32 located at the 3' end of the first flanked reverse primer 30, and a non-specific flank section 34 located at the 5' end of the first flanked reverse primer 30. The flank section 24 of the first flanked forward primer 20 is identical to the binding section 12 of the superprimer 10. The flank section 34 of the first flanked reverse primer 30 has a recognition sequence for the first nicking enzyme. In a different embodiment, the flank section 34 of the first flanked reverse primer 30 may have a recognition sequence for a second nicking enzyme.

[0127] Figure 3B shows the structure of a pair of flanked primers 120, 130, of which variant (ii) is present. The pair comprises a flanked forward primer 120 and a flanked reverse primer 130. The flanked forward primer 120 has two sections 122, 124: a target-specific section 122, located at the 3' end of the flanked forward primer 120, and a non-specific flank section 124, located at the 5' end of the flanked forward primer 120. The flanked reverse primer 130 has two sections 132 and 134: a target-specific section 132 located at the 3' end of the flanked reverse primer 130, and a non-specific flank section 134 located at the 5' end of the flanked reverse primer 130. The flank section 124 of the flanked forward primer 120 has a recognition sequence for the first nicking enzyme.The flank section 134 of the flanked reverse primer 130 is identical to the bonding section 12 of the superprimer 10. In contrast to the present embodiment, the flank section 124 of the flanked forward primer 120 may have a recognition sequence for a second nicking enzyme.

[0128] Figure 3C shows the structure of a pair of flanked primers 220, 230, of which variant (iii) is present. The flanked forward primer 220 has two sections 222, 224: a target-specific section 222 located at the 3' end of the flanked forward primer 220, and a non-specific flank section 224 located at the 5' end of the flanked forward primer 220. The flanked reverse primer 230 has two sections 232, 234: a target-specific section 232 located at the 3' end of the flanked reverse primer 230, and a non-specific flank section 234 located at the 5' end of the flanked reverse primer 230. The flank section 224 of the flanked forward primer 220 is identical to the binding section 12 of the superprimer 10. Likewise, the flank section 234 of the flanked reverse primer 230 is identical to the binding section 12 of the superprimer 10.

[0129] In the present embodiment, variant (i) is present for all three pairs of flanked primers. Figure 3A shows the structure of the first pair of flanked primers 20, 30, which also applies accordingly to the second and third pairs of flanked primers. In contrast to the present embodiment, variant (ii) or variant (iii) can be present for all pairs of flanked primers. Furthermore, different variants can be present for different pairs of flanked primers, since the presence of one of variants (i), (ii), or (iii) applies individually to each pair of flanked primers. For example, variant (i) can be present for a first pair of flanked primers and variant (ii) for a second pair of flanked primers.

[0130] The recognition section 14 of the superprimer 10 and the flank section 34 of the first flanked reverse primer 30 differ except for the recognition sequence for the first nicking enzyme. This applies accordingly to the flank section of the second flanked reverse primer and the flank section of the third flanked reverse primer, which in the present embodiment are identical to the flank section 34 of the first flanked reverse primer 30.

[0131] By using the superprimer 10, the concentrations of the flanked primers and the external primers can be reduced so significantly that unwanted interactions between the primers occur only to a minimal extent or not at all. This enables the method to efficiently amplify the majority of target nucleic acid sequences.

[0132] In the present embodiment, the superprimer 10 is used at a higher concentration than the flanked primers. This promotes the progression of SDA, since SDA is primarily driven by the superprimer. The external primers are used at a lower concentration than the flanked primers. The concentration of the external primers is the same for all external primers. In contrast to the present embodiment, the concentration of the external primers can differ between different external primers.

[0133] The flanked backward primers are each used in a concentration that is 0.5 times higher than the concentration of the corresponding flanked forward primer. This promotes, right from the start of SDA, that for each target nucleic acid sequence, the strand of the target nucleic acid sequence to which the flanked forward primer binds, at least at the beginning of SDA, is preferentially amplified.

[0134] The concentration of the flanked forward and reverse primers is not the same for all pairs of flanked primers. In contrast to the present embodiment, the concentration of the flanked forward and reverse primers can be the same for all pairs of flanked primers.

[0135] Specifically, the following concentrations are used in the present embodiment:

[0136] Superprimer 10 1,40 ppm

[0137] First flanked forward primer 20 0.08 pM First flanked backward primer 30 0.04 pM First external forward primer 40 0.02 pM First external backward primer 50 0.02 pM Second flanked forward primer 0.08 pM Second flanked backward primer 0.04 pM Second external forward primer 0.02 pM Second external backward primer 0.02 pM Third flanked forward primer 0.06 pM Third flanked backward primer 0.03 pM

[0138] Third external forward primer 0.02 pM Third external backward primer 0.02 pM

[0139] In the present embodiment, the length of the flanked primers, with the exception of the third flanked forward primer, is 40 nucleotides each. The length of the third flanked forward primer is 41 nucleotides. The length of the external primers is 20 nucleotides each, and the length of the superprimer is 40 nucleotides.

[0140] SDA is performed using the first nicking enzyme. In the present embodiment, only this nicking enzyme is used. In this embodiment, Nt.BspQI from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA) is used as the first nicking enzyme.

[0141] SDA is performed using three detection probes (one probe per target nucleic acid sequence to be amplified): a first probe specific for the first target nucleic acid sequence, a second probe specific for the second target nucleic acid sequence, and a third probe specific for the third target nucleic acid sequence. Each detection probe generates its own specific fluorescence signal. The detection probes are designed as molecular beacons. These probes enable the detection of the amplification products obtained in step S300. Thus, SDA is performed using real-time detection of the amplification products.In contrast to the present embodiment, the amplification products obtained in step S300 can be detected by a downstream detection method or otherwise reused. Prior to this, the amplification products obtained in step S300 can be cooled or allowed to cool.

[0142] The detection probes each bind to the strand of the corresponding target nucleic acid sequence that is preferentially amplified in SDA. For pairs of flanked primers, where variant (i) is present, this is the strand to which the flanked forward primer binds, at least initially, during SDA.

[0143] In the present embodiment, the concentration of the detection probes is in the range of 0.20 to 0.40 pM.

[0144] For SDA, a DNA polymerase with strand displacement activity, specifically the SD polymerase from Bioron GmbH (Bioron GmbH, Römerberg, Germany), and nucleotides (dNTPs) of the types deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) are used. SDA is performed in a suitable buffer compatible with the DNA polymerase. In the present embodiment, SDA is carried out in a reaction volume of 20 pl for a duration of 20 minutes.

[0145] The present embodiment relates to a diagnostic test. The first target nucleic acid sequence is a nucleic acid sequence of Chlamydia trachomatis (CT), and the second target nucleic acid sequence is a nucleic acid sequence of Neissera gonorrhoeae (NG). CT and NG are among the most common sexually transmitted pathogens. The diagnostic test serves to detect the presence of CT and NG in patient samples and can be performed point-of-care or as an on-site analysis. The third target nucleic acid sequence is a nucleic acid sequence of human beta-actin and serves as a control. This control ensures that sexually transmitted infection (SDA) has actually occurred. Furthermore, in this embodiment, the control ensures that the sample material was collected correctly, including cellular material (thus preventing false-negative results due to incomplete sample collection).

[0146] The third flanked forward primer has an additional segment at the 5' end of its flank region. The sequence of this additional segment is identical to the portion of the recognition region 14 of the superprimer 10 following binding region 12, corresponding to the length of the additional segment. The length of the additional segment is 1 nucleotide. Since the third target nucleic acid sequence serves only as a control, it is sufficient that it is amplified less strongly among the majority of target nucleic acid sequences.

[0147] In this method, the three target nucleic acid sequences are amplified simultaneously. For the sake of simplicity, however, the following description of the first amplification and reaction steps refers only to the amplification of the first target nucleic acid sequence. The first target nucleic acid sequence has a first strand 60 and a complementary second strand 60'. The first strand 60 and the second strand 60' are separated in step S200. This occurs in the presence of the first pair of flanked primers 20, 30, the first pair of external primers 40, 50, and the superprimer 10. In contrast to the present embodiment, the first detection probe may also be present in step S200.SDA begins, as schematically depicted in Figure 4, with the binding of the first external forward primer 40 and the target-specific region 22 of the first flanked forward primer 20 to the complementary regions of the first strand 60, and with the binding of the first external reverse primer 50 and the target-specific region 32 of the first flanked reverse primer 30 to the complementary regions of the second strand 60'. The DNA polymerase continuously adds single nucleotides 5' to 3' towards the first flanked forward primer 20 and the first flanked reverse primer 30. This is indicated in Figure 4 by the arrows with the dashed lines. This first reaction step results in the formation of two newly synthesized DNA strands, one of which is complementary to the first strand 60 and has the flank section 24 of the first flanked forward primer 20 at its 5' end.In a second reaction step, the target-specific section 32 of the first flanked reverse primer 30 binds to this newly synthesized strand. The DNA polymerase again continuously adds single nucleotides to the first flanked reverse primer 30, resulting in another newly synthesized strand that has the flank section 34 of the first flanked reverse primer 30 at its 5' end. This strand, newly synthesized in the second reaction step, has a section complementary to the flank section 24 of the first flanked forward primer 20, to which the binding site 12 of the superprimer 10 binds in a third reaction step. The superprimer 10 is extended by the DNA polymerase, resulting in a newly synthesized DNA strand that has the recognition section 14 of the superprimer 10 at its 5' end.A fourth reaction step follows, after which the recognition region 14 of the superprimer 10 is double-stranded, leading to a single-strand break caused by the first nicking enzyme. At the site of the single-strand break, the DNA polymerase can bind again and synthesize the complementary strand, while the already present complementary strand is displaced. In this way, another strand is formed that is complementary to the first strand 60. Thus, the superprimer 10 promotes the amplification of the first strand 60, to which the first flanked forward primer 20 bound at the beginning of SDA. This results in the first strand 60 being preferentially amplified over the second strand 60'. The first detection probe binds to the first strand 60 and its amplification products. This leads to a fluorescence signal that is specific for the first detection probe and is optically detected in real time.The other strand newly synthesized in the first reaction step is complementary to the second strand 60' and has the flank region 34 of the first flanked reverse primer 30 at its 5' end, thus carrying the recognition sequence for the first nicking enzyme. In the second reaction step, the target-specific region 22 of the first flanked forward primer 20 binds to this newly synthesized strand. The DNA polymerase again continuously adds single nucleotides to the first flanked forward primer 20, resulting in another newly synthesized strand that has the flank region 24 of the first flanked forward primer 20 at its 5' end. Due to the strand newly synthesized in the second reaction step, the recognition sequence for the first nicking enzyme is now present in a double strand, leading to a single-strand break by the first nicking enzyme.At the site of the single-strand break, DNA polymerase can bind again and synthesize the complementary strand, while the already existing complementary strand is displaced. In this way, another strand is created that is 60' complementary to the second strand.

[0148] Through repeated nicking and strand synthesis, an exponential amplification of the first target nucleic acid sequence takes place.

[0149] The description of the first amplification or reaction steps for the first target nucleic acid sequence applies accordingly to the first amplification steps of the second target nucleic acid sequence and the first amplification steps of the third target nucleic acid sequence. This also applies to the description of the detection of the corresponding amplification products.

[0150] In contrast to the present embodiment, the method can also be used for target nucleic acid sequences that are in the form of RNA, in which the target nucleic acid sequences that are in the form of RNA are additionally transcribed into complementary DNA (cDNA) by means of reverse transcription.

Claims

Patent claims 1. Method for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample, the method comprising: (S300) Amplification of the target nucleic acid sequences by isothermal strand displacement amplification using pairs of flanked primers, using a superprimer (10) and using at least one first nicking enzyme, wherein one pair of flanked primers is used for each target nucleic acid sequence to be amplified and the pair of flanked primers is specific for the associated target nucleic acid sequence, wherein each pair of flanked primers has a flanked forward primer and a flanked reverse primer, wherein each flanked forward primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, wherein the target-specific region is complementary to a region of the associated target nucleic acid sequence.wherein each flanked reverse primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, wherein the target-specific region is complementary to a region of the associated target nucleic acid sequence, wherein the superprimer (10) has a binding region (12) at its 3' end and a recognition region (14) at its 5' end, wherein the recognition region (14) has a recognition sequence for the first nicking enzyme, wherein for each pair of flanked primers individually one of the following variants is present:, (i) the flank section of the flanked forward primer is identical to the binding section (12) of the superprimer (10), and the flank section of the flanked reverse primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, (ii) the flank section of the flanked forward primer has a recognition sequence for the first nicking enzyme or for a second Nicking enzyme, and the flank section of the flanked reverse primer is identical to the binding section (12) of the superprimer (10), (iii) the flank section of the flanked forward primer is identical to the bonding section (12) of the superprimer (10) and the flank section of the flanked backward primer is identical to the bonding section (12) of the superprimer (10).

2. Method according to claim 1, wherein variant (i) or variant (ii) is individually available for each pair of flanked primers.

3. Method according to claim 2, wherein variant (i) is present for all pairs of flanked primers or variant (ii) is present for all pairs of flanked primers.

4. Method according to one of claims 1 to 3, wherein in step (S300) additionally a detection of the amplification products obtained in this step is carried out.

5. Method according to any one of claims 1 to 4, further comprising: (S200) Heating the sample to separate double-stranded target nucleic acid sequences into their single strands.

6. A method according to any one of claims 1 to 5, wherein step (S300) is additionally carried out using pairs of external primers, wherein one pair of external primers is used for each target nucleic acid sequence to be amplified, and the pair of external primers is specific for the associated target nucleic acid sequence, wherein each pair of external primers comprises an external forward primer and an external reverse primer, wherein the external forward primer is complementary to a region of the associated target nucleic acid sequence that lies 3'-ward to the region to which the sequence-specific region of the flanked forward primer associated with that target nucleic acid sequence is complementary, and wherein the external reverse primer is complementary to a region of the associated target nucleic acid sequence that lies 3'-ward to the region to which the sequence-specific section of the flanked reverse primer belonging to this target nucleic acid sequence is complementary.

7. Method according to any one of claims 1 to 6, wherein the superprimer (10) is used in a higher concentration than the flanked primers.

8. Method according to claim 7, wherein the superprimer (10) is used in a concentration that is about 15 to 25 times higher than the concentration of one of the flanked primers that is used in the highest concentration among the flanked primers.

9. Method according to any one of claims 1 to 8, wherein in variant (i) the flanked reverse primer is used in a concentration that is about 0.5 to 0.75 times higher than the concentration of the flanked forward primer.

10. Method according to any one of claims 1 to 9, wherein in variant (ii) the flanked forward primer is used in a concentration that is about 0.5 to 0.75 times higher than the concentration of the flanked reverse primer.

11. Method according to any one of claims 1 to 10, wherein in at least one, but not in all pairs of flanked primers, an additional section is arranged at the 5' end of the flank section which is identical to the bonding section (12) of the superprimer (10), wherein the sequence of the additional section is identical to the part of the recognition section (14) of the superprimer (10) following the bonding section (12) according to the length of the additional section.

12. Primer set for the simultaneous amplification of multiple target nucleic acid sequences in a sample, the primer set comprising: (a) pairs of flanked primers, and (b) a superprimer (10) wherein one pair of flanked primers is provided for each target nucleic acid sequence to be amplified and the pair of flanked primers for the assigned- a target nucleic acid sequence is specific, wherein each pair of flanked primers comprises a flanked forward primer and a flanked reverse primer, wherein each flanked forward primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the associated target nucleic acid sequence, wherein each flanked reverse primer has a target-specific region at its 3' end and a non-specific flank region at its 5' end, the target-specific region being complementary to a region of the associated target nucleic acid sequence, wherein the superprimer (10) has a binding region (12) at its 3' end and a recognition region (14) at its 5' end, the recognition region (14) having a recognition sequence for a first nicking enzyme,where each pair of flanked primers individually exhibits one of the following variants: (i) the flank section of the flanked forward primer is identical to the binding section (12) of the superprimer (10), and the flank section of the flanked reverse primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, (ii) the flank section of the flanked forward primer has a recognition sequence for the first nicking enzyme or for a second nicking enzyme, and the flank section of the flanked reverse primer is identical to the binding section (12) of the superprimer (10), (iii) the flank section of the flanked forward primer is identical to the bonding section (12) of the superprimer (10) and the flank section of the flanked backward primer is identical to the bonding section (12) of the superprimer (10).

13. Use of the primer set according to claim 12 for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample.

14. Kit for the simultaneous amplification of multiple target nucleic acid sequences in a sample, the kit comprising: (a) Primer set according to claim 12, and (b) the first nicking enzyme and / or the second nicking enzyme.

15. Use of the kit according to claim 14 for the simultaneous amplification of a plurality of target nucleic acid sequences in a sample.

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