Nucleic Acid Detection

Abstract

A method for detecting at least one target nucleic acid sequence in a sample, comprising adding a sequence for an RNA polymerase to the target nucleic acid and detecting protons released by, for example, the transcription activity of the RNA polymerase.

Description

[Technical field]

[0001] The present invention relates to a method for detecting at least one target nucleic acid sequence in a sample, which comprises adding a sequence for an RNA polymerase to the target nucleic acid and detecting protons released by, for example, the transcription activity of the RNA polymerase. [Background technology]

[0002] Quantitative real-time polymerase chain reaction (qPCR) has become the standard for amplifying small amounts of DNA or RNA biomarkers. Most embodiments of qPCR and nucleic acid detection require fluorescently labeled sequence-specific probes or intercalating dyes. Exponential production of PCR products causes a concomitant exponential increase in fluorescence. In multiplex PCR, multiple target biomarker sequences within the same sample can be amplified in a qPCR reaction by including multiple primer pairs and different fluorescently labeled sequence-specific probes that can be detected simultaneously or sequentially (e.g., using optical filters). However, the potential for highly multiplexed PCR methods is hindered by the unavoidable spectral overlap between fluorescent dyes and the limited segments of the spectrum in affordable dyes for water-based solutions. In qPCR, the ability to multiplex targets using differently labeled probes is typically limited to the analysis of five target sequences in a single qPCR reaction.

[0003] Currently, the field of biochemical analysis is undergoing a miniaturization effort with great attention to developing portable and compact systems, often using microfluidics to integrate tasks that previously required entire laboratories. Such microfluidic devices are usually aimed at handling minute amounts of liquids and analytes, improving multiplexing capabilities, and enabling high throughput, fast processing times, and low costs in a very small footprint for performing biomolecular techniques.

[0004] Monitoring biological events such as nucleic acid hybridization using label-free biosensing methods based on field-effect transistors (FETs) has attracted considerable attention, especially due to their potential for miniaturization, easy integration at low cost into standard CMOS technology and the required electronics, and multiplexing capabilities. FET-based methods rely on electrical signals rather than optical signals (electrochemical detection) and therefore are not subject to the limitations imposed by fluorescent dyes and bulky equipment.

[0005] Ion-sensitive field-effect transistors (ISFETs), sometimes simply called pH sensors, measure the concentration of ions in solution. Their suitability as readout in amplification reactions has been widely studied.

[0006] WO 2003 / 073088 discloses a thermally actuated complementary metal oxide semiconductor (CMOS) based amplification device integrated with reaction chemistry. ISFETs have been used to monitor protons released in the pyrophosphate hydrolysis reaction associated with the insertion of individual nucleotides at the ends of oligonucleotide chains. Detection of individual nucleotide insertions by pH-sensitive ISFETs has been used in DNA sequencing techniques based on "decoding-by-synthesis".

[0007] WO 2008 / 107014 also relies on protons being the product of PCR. Therefore, qPCR can also be achieved by using a pH-sensitive ISFET to monitor the protons released with the insertion of each nucleotide as the amplification proceeds. Amplification is monitored by detecting changes in pH. The sensitivity required for detection is achieved by carrying out the amplification at low volumes and low buffer capacity, ensuring that the released protons cause a rapid change in pH when the buffer capacity of the sample is exceeded. The use of a DNA probe immobilized on an ISFET to capture the target DNA and separate it from unwanted interfering products is described.

[0008] Fabrication of arrays using scaled-down ISFET designs has enabled many sensors to be integrated onto a miniaturized chip with little effort. For nucleic acid applications, large FET arrays for monitoring biological events have been described, particularly for use in nucleic acid sequencing.

[0009] WO 2010 / 138182 describes methods and devices for FET arrays, including large FET arrays, for monitoring chemical and / or biological reactions, such as nucleic acid sequencing reactions. Some of the methods described therein relate to improving the signal (and signal to noise ratio) of hydrogen ions released during nucleic acid sequencing reactions.

[0010] WO 2010 / 047804 is directed to devices and chips with an array of large-scale chemical field effect transistors having an array of sample retention areas capable of retaining chemical or biological samples from a sample fluid for analysis, the devices and chips having large-scale pH-based DNA sequencing and other bioscience and biomedical applications.

[0011] In addition to applications in nucleic acid sequencing, the utility of ISFETs for monitoring nucleic acid amplification has also been described as a CMOS chip platform incorporating loop-mediated isothermal amplification (LAMP) and PCR.

[0012] Thus, Toumazou et al. (2013, Nat Methods 10:641-646) used standard CMOS process flows to fabricate an integrated circuit that amplifies and simultaneously detects DNA on a chip with an embedded heater, 10 temperature sensors, and 40 ISFET sensors. LAMP and PCR conditions were optimized with low buffer conditions that maintained amplification efficiency and specificity. Multiplexing capabilities were demonstrated by simultaneously interrogating two known biomarkers.

[0013] Duarte-Guevara et al. (2014, Anal Chem 86:8359-8367) investigated improved biosensing resolution for LAMP reactions using foundry-fabricated individually addressable dual-gate ISFETs.

[0014] Furthermore, the applicability of the ISFET chip architecture by Toumazou and Duarte-Guevara (op. cit.) was tested in the clinic (Duarte-Guevara et al (2016), RSC Adv 6:103872-103887), where a dual-gate ISFET array platform was used for on-chip electrical detection of LAMP reactions targeting food-borne bacterial pathogens.

[0015] Although the cited publications describe ISFET methods as viable biosensing techniques that are amenable to automation and offer many advantages, these methods still suffer from drawbacks that limit their practical application.

[0016] One such drawback is that electrochemical detection in PCR requires a relatively long time for the amplification reaction to generate a sufficient amount of protons to obtain a measurable signal, especially when the target nucleic acid is in low concentration. In this regard, Tomazou et al. (op. cit.) state that 40 cycles of on-chip pH measurement PCR require 35 minutes to complete, which does not reduce the turnaround time of conventional optically based PCR devices. In certain settings, e.g. point-of-care diagnostics, it is necessary to obtain test results as quickly as possible without losing sensitivity, as well as a portable and compact system, to allow for rapid intervention in patient management and patient outcomes. Furthermore, detection using ISFETs exposed to thermal cycling (e.g. in PCR) may be difficult, since the temperature sensitivity of the ISFET must always be taken into account and compensated for.

[0017] Furthermore, current approaches to PCR using ISFET as a detection method often involve highly specific assays targeting specific analytes, which require the creation of multiple microfluidic chambers into each of which a different primer is injected, increasing the footprint in highly multiplexed settings, or the chip cannot be used if a new target needs to be included in the assay, as the primers are directly attached to the chip to initiate the reaction. In this case, every time a new target needs to be added, a new chip must be manufactured containing the probe for the new target, which is very labor intensive in terms of manufacturing. This is particularly relevant for patient screening, where an all-in-one diagnostic approach is desirable (screening multiple analytes on one chip).

[0018] There remains a need for alternative nucleic acid analysis techniques. Summary of the Invention

[0019] One objective of this disclosure is to overcome the limitations of conventional biosensor detection methods.

[0020] Another object of this disclosure is to provide a method for generating sufficient amounts of protons to reliably detect target nucleic acids.

[0021] Another object of this disclosure is to overcome the limitations of conventional ISFET-based detection methods.

[0022] Another object of this disclosure is to enable the use of ISFET sensors without the need for high temperature conditions that would affect the sensitivity and / or reliability of the test.

[0023] It is yet another object of this disclosure to provide methods for detecting target nucleic acid sequences in a manner that is amenable to miniaturized and / or portable systems.

[0024] Yet another object of this disclosure is to provide a rapid turnaround method to speed up existing techniques for detecting target nucleic acid sequences, for example in diagnostic or prognostic determinations.

[0025] These objectives, as well as other objectives apparent to those skilled in the art from the teachings of this specification, are met by various aspects of this disclosure as defined in the specification and claims.

[0026] Thus, in a first aspect, the disclosure provides a method for detecting the presence of at least one target nucleic acid sequence in a sample, comprising: - providing a sample suspected of containing said at least one target nucleic acid sequence; - adding a promoter sequence for an RNA polymerase to a target nucleic acid sequence present in said sample; - said sample - at least one detection zone; and - at least one capture nucleic acid disposed on a solid support and adapted to indirectly or directly bind to said target nucleic acid; into a reaction chamber having generating a sequence of single-stranded nucleic acid bound directly or indirectly to the capture nucleic acid disposed on the solid support; - applying extension conditions that allow the generation of a nucleic acid strand complementary to said single-stranded nucleic acid to form a double-stranded nucleic acid that contains a promoter sequence for an RNA polymerase and is directly or indirectly bound to said capture nucleic acid disposed on said solid support; - applying transcription conditions that allow the production of transcripts from the double-stranded nucleic acid captured on the solid support, the production of transcripts releasing protons as transcription proceeds; and - detecting the presence of said protons as a signal from said detection zone, said signal being indicative of the presence of said target nucleic acid sequence in said sample; The method includes the steps of:

[0027] As used herein, the term "biological sample" or simply "sample" is intended to mean one or more of a variety of biological sources containing nucleic acids and / or cellular material, whether freshly obtained from an organism (i.e., a fresh tissue sample) or preserved by methods known in the art (e.g., an FFPE sample). Examples of samples include cell cultures, such as mammalian cells or eukaryotic microorganisms; body fluids; body fluid sediments; lavage samples; fine needle aspirates; biopsy samples; tissue samples; cancer cells; other cells obtained from a patient; cells derived from tissues or cultured in vitro from a subject being tested and / or treated for disease or infection; or forensic samples. Examples of body fluids include whole blood, bone marrow, cerebrospinal fluid (CSF), peritoneal fluid, pleural fluid, lymph, serum, plasma, urine, chyle, stool, sperm, sputum, nipple aspirate, saliva, swab samples, wash / lavage fluid, and / or brush samples.

[0028] In this specification, the terms "nucleic acid" and "its equivalent polynucleotide" refer to a polymer of ribonucleotides or deoxyribonucleotides linked by phosphodiester bonds between the nucleotide monomers. The sequence of these bases (or nucleosides or nucleotides) in a nucleic acid chain is called a "nucleic acid sequence" and is indicated in the so-called 5' to 3' direction, which refers to the chemical direction of the nucleic acid chain. A sample suspected of containing at least one target nucleic acid sequence is a sample suspected of containing a target nucleic acid having a target nucleic acid sequence. Nucleic acids include, but are not limited to, DNA and RNA, including genomic DNA, mitochondrial or meDNA, cDNA, mRNA, rRNA, tRNA, hnRNA, microRNA, lncRNA, siRNA, and various modifications thereof. Nucleic acids are most commonly obtained from natural sources, such as biological samples obtained from various types of organisms. Nucleic acids can also be synthetic, recombinant, or produced or manipulated by known methods (e.g., PCR).

[0029] Regardless of whether the target nucleic acid is directly or indirectly bound to the capture nucleic acid, the target nucleic acid is suitably introduced into the reaction chamber in single-stranded form. Methods for obtaining single-stranded nucleic acid from double-stranded nucleic acid are well known to those skilled in the art and may include, for example, heating the double-stranded nucleic acid at a temperature high enough to denature the double-stranded nucleic acid (e.g. 90° C.) or may include chemical treatment of the double-stranded target nucleic acid. Such treatment methods (denaturing conditions) may be applied before the sample is introduced into the reaction chamber or may be applied in the reaction chamber before the resulting single-stranded target nucleic acid is hybridized to the capture nucleic acid.

[0030] In one embodiment, when the target nucleic acid is introduced, the temperature of the reaction mixture containing the target nucleic acid is 75-80° C. At this temperature, the single-stranded target nucleic acid hybridizes directly or indirectly to the capture nucleic acid. The product of the hybridization of the target nucleic acid with the capture nucleic acid serves as a substrate for extension conditions, in which the single-stranded portion of the target nucleic acid is "buried" to produce double-stranded DNA of the entire target nucleic acid sequence and a functional double-stranded RNA polymerase protomer sequence.

[0031] The "capture nucleic acid" disposed on a solid support suitable for use in the present invention can be selected from the group consisting of, for example, DNA, RNA, PNA (peptide nucleic acid), LNA (locked nucleic acid), ANA (arabino nucleic acid) and HNA (hexitol nucleic acid). It may be an oligonucleotide that allows the formation of a homoduplex (DNA:DNA) or heteroduplex with the target nucleic acid under suitable hybridization conditions. As shown in the methods described herein, at least one single-stranded nucleic acid sequence is directly or indirectly bound to the capture nucleic acid disposed on the solid support. In this specification, the terms "bound" and "binding" are used interchangeably with "hybridized" to refer to the direct or indirect capture of the target nucleic acid to allow surface-specific detection in the vicinity of the capture nucleic acid on the solid support. The capture portion of the capture nucleic acid (also called capture probe) may comprise 10 to 200 nucleotides, preferably 15 to 50 nucleotides, specific for the target or adapter nucleic acid sequence to which it is bound. Ideally, the capture nucleic acid comprises 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides. The capture nucleic acid may also comprise additional nucleotides that can act as spacers between the capture moiety and the solid surface or have a stabilizing function. The number of such additional nucleotides may be 0 to 200, preferably 0 to 50. Ideally, the spacer nucleic acid consists of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides.

[0032] "Adding an RNA polymerase sequence to a target nucleic acid sequence present in a sample" means that a promoter sequence for an RNA polymerase is incorporated into the target nucleic acid present in the sample. A variety of enzymatic methods suitable for this purpose are widely known, including, for example, ligation techniques, recombination techniques, and PCR (polymerase chain reaction).

[0033] By "RNA polymerase promoter sequence", one of skill in the art will recognize that this is a nucleotide sequence that is selectively recognized by an RNA polymerase and acts as a promoter of the binding and activity of that enzyme. It is within the ability of one of skill in the art to select and / or design such a sequence suitable for use with a given specific RNA polymerase enzyme. In one embodiment, the length of the RNA polymerase promoter sequence is 17-26 base pairs. One example of a suitable RNA polymerase promoter sequence is the T7 RNA polymerase promoter sequence, which comprises 5'-TAATACGACTCACTATA-3' (SEQ ID NO: 1). In some cases, it may be preferable for an efficient promoter to comprise at least one or more G nucleotide bases. Thus, another sequence used for transcription of T7 RNA may comprise 5'-TAATACGACTCACTATAG-3' (SEQ ID NO: 2).

[0034] After the double-stranded DNA is generated under extension conditions, new conditions (transcription conditions) are applied to allow the RNA polymerase to transcribe from the promoter sequence. In other words, under these conditions, the RNA molecule is synthesized in a repeated initiation cycle, releasing one proton for each new incorporation of a nucleotide. The fast isothermal reaction causes a very short-term local change in pH, which can be measured in the detection zone, e.g., in the detection unit.

[0035] Thus, in the method of the first aspect, the target nucleic acid is provided with the promoter sequence of RNA polymerase and immobilized via capture nucleic acid on a solid support in a reaction chamber.After the target nucleic acid is converted into a double-stranded form, its presence can be detected by transcription, applying transcription conditions.During transcription, protons are released and detected in a detection zone provided in the reaction chamber.

[0036] Thus, the method of the first aspect has the advantage that it allows detection of the target nucleic acid separately from other steps, such as the amplification step of PCR. One consequence of this is that detection can be carried out under isothermal conditions, e.g. at a relatively low, constant temperature, such as room temperature, rather than at high temperatures that involve thermal cycling.

[0037] Another advantage of the method of the first aspect is that the transcription conditions can be adapted such that a large amount of protons are released in a short time, ensuring efficient and reliable detection in the detection zone. When NTPs are incorporated during transcription, protons may be released directly or indirectly and simultaneously via conversion of released pyrophosphate.

[0038] Yet another advantage of the method is that it is suitable for processing small volumes of liquid and analytes.Another advantage is that the method is suitable for adaptation to multiplex formats, as described below.

[0039] In one embodiment of the method, the detection zone comprises a detection unit that detects protons, i.e. measures the change in pH. There are various suitable techniques in the art for measuring pH changes and thus detecting protons. The pH change may be measured, for example, using pH indicators, such as fluorescence, or the absorbance of the solution. pH indicators that change the color of the solution depending on the pH and optical detection units that measure the pH change are well known to those skilled in the art. In a specific embodiment, the detection unit is an optical system in the reader. In another specific and advantageous embodiment, the detection unit is an ion-sensitive field effect transistor (ISFET).

[0040] In certain embodiments, the extension conditions for converting a single-stranded target nucleic acid into a double-stranded nucleic acid include the presence of a DNA polymerase.

[0041] In one embodiment, the extension conditions include a reaction temperature of 75-90°C.

[0042] In certain embodiments, the transcription conditions include the presence of an RNA polymerase. In more specific embodiments, the RNA polymerase is the publicly available T7 RNA polymerase.

[0043] In one embodiment, the transfer conditions include a reaction temperature of 20 to 40° C. The reaction temperature of the transfer conditions may be advantageously near room temperature for optimal performance and durability of a detection unit, such as an ISFET, disposed in the detection zone.

[0044] In certain embodiments, the method of the first aspect of this disclosure is a method for detecting the presence of multiple target nucleic acid sequences, useful, for example, for monitoring several biomarkers that may be present in a patient sample. In such embodiments, the multiple target sequences correspond to multiple capture nucleic acids disposed on a solid support, each of the capture nucleic acids adapted to bind to a different target nucleic acid sequence.

[0045] In a more specific form of this embodiment, the plurality of capture nucleic acids are arranged in the form of an array on the solid support, with each capture nucleic acid representing an addressable location on said array. In such a setup, the detection zone may in one embodiment be adapted to identify the location on the array at which a signal is detected.

[0046] In one embodiment of the method of the first aspect of this disclosure, the or each capture nucleic acid is designed such that its sequence matches the sequence of the target nucleic acid sequence it is intended to capture. In this embodiment, at least a portion of the sequence of the or each capture nucleic acid is identical to or complementary to at least a portion of the target nucleic acid sequence and directly binds to one of the strands of the double-stranded nucleic acid to be detected.

[0047] FIG. 1 shows one embodiment of the method of the first aspect of this disclosure, in which two capture nucleic acids of different sequences are immobilized on two array addresses i and j of a solid support in a reaction chamber. Each capture nucleic acid is designed such that its sequence or a portion of its sequence matches (complements) a portion of the sequence of the target nucleic acid to be captured. As shown in FIG. 1 (1) ("(1)" is a circle on a 1, the same applies below), a single-stranded target nucleic acid to which a promoter sequence for an RNA polymerase is added (incorporated) hybridizes with the target-specific sequence of capture nucleic acid 1 and is immobilized at address i of the solid support. As shown in (2), extension conditions are applied such that a double-stranded nucleic acid containing an RNA polymerase sequence is formed using the single-stranded target nucleic acid as a template for the generation of a complementary strand. Finally, as shown in (3), in this embodiment involving an RNA polymerase, transcription conditions are applied to produce several transcripts from the immobilized double-stranded target sequence, leading to an associated release of protons that are detected in the detection zone, for example by a detection unit such as an ISFET.

[0048] In another advantageous embodiment, at least one capture nucleic acid, e.g., a capture nucleic acid present at a predetermined address on an addressable support, instead comprises a unique adapter sequence that is not derived from the target, and the method includes the use of an adapter nucleic acid sequence to avoid the need to tailor the solid support to the predetermined target nucleic acid to be detected. In this embodiment: - the step of adding a promoter sequence for an RNA polymerase further comprises the step of adding a specific adapter sequence to a target nucleic acid sequence present in the sample; - the or each capture nucleic acid comprises a unique adapter sequence; and The reaction chamber comprises: - a first specific adapter sequence that is identical or complementary to a specific adapter sequence in a target nucleic acid sequence present in the sample; and - a second unique adapter sequence that is complementary to the unique adapter sequence of the capture nucleic acid; and further comprising at least one adaptor nucleic acid comprising:

[0049] In this embodiment, at least one capture nucleic acid present at a given address in the addressable solid support is unique in the sense that it does not directly match the sequence of the target nucleic acid, nor is it identical to a corresponding unique adapter sequence at another address, e.g., in an array.

[0050] In one embodiment, the specific adapter nucleic acid sequence added to the target nucleic acid sequence present in the sample may consist of at least a part of a nucleic acid sequence already present in the target nucleic acid. This is the case, for example, when the specific adapter sequence is added by an amplification reaction, for example PCR. In this case, the adapter nucleic acid sequence is identical or complementary to the nucleic acid sequence of one of the primers used in the amplification reaction, the same primer nucleic acid sequence being identical or complementary to a part of the target nucleic acid sequence.

[0051] In embodiments using an adapter nucleic acid in the reaction chamber, at least a portion of the specific adapter sequence of the adapter nucleic acid is identical or complementary to at least a portion of a corresponding nucleic acid sequence in the target nucleic acid present, and at least another unique portion of the nucleic acid sequence of the adapter nucleic acid is identical or complementary to at least a portion of the capture nucleic acid sequence and directly binds to one of the strands of the double-stranded nucleic acid to be detected.

[0052] This design of the capture and adapter sequences allows the or each capture nucleic acid to indirectly bind to the corresponding target nucleic acid to be detected by overlapping hybridization of the or each adapter nucleic acid with both the capture nucleic acid and the or each target nucleic acid. In this embodiment, by designing appropriate adapter sequences, it is possible to limit the production to only one type of solid support (e.g. chip) that can be tailored to detect all possible target nucleic acid sequences.

[0053] FIG. 2 illustrates one embodiment of the method of the first aspect of this disclosure, in which capture nucleic acids of various sequences are immobilized on two array addresses i and j of a solid support in a reaction chamber. Two different adaptor nucleic acids are present in the reaction chamber, each with a different unique adaptor sequence that hybridizes to a corresponding capture nucleic acid sequence at a specific address, each with a different target-specific sequence. Adaptor nucleic acid 1 in FIG. 2 is specific to target nucleic acid 1, while adaptor nucleic acid 2 has a specific sequence that is specific to another target nucleic acid not present in the reaction chamber, as shown. As shown in (1) of FIG. 2, a single-stranded target nucleic acid with an RNA polymerase promoter sequence added is added, hybridized to the target-specific sequence of adaptor nucleic acid 1, and immobilized at address i of the solid support. As shown in (2), extension conditions are applied to form a double-stranded nucleic acid containing an RNA polymerase sequence, using the single-stranded target nucleic acid as a template for the generation of a complementary strand. Finally, as shown in (3), in this embodiment comprising an RNA polymerase, transcription conditions are applied, producing several transcripts from the immobilized double-stranded target sequence, leading to an associated release of protons that are detected in a detection zone, for example by a detection unit such as an ISFET.

[0054] As previously described, the method according to the first aspect of this disclosure comprises adding an RNA polymerase promoter sequence to a target nucleic acid sequence present in a sample to be analyzed. In certain aspects of the disclosed method, this addition of the RNA polymerase promoter sequence is performed as part of an amplification reaction. Suitably, the amplification reaction is designed to amplify the target sequence present in a patient sample or other sample of genetic material. This aspect is thus similar to known amplification methods for detecting biomarkers or other sequence variants in a sample of genetic material, with the advantageous difference that the initial target amplification reaction is separate from the subsequent steps of target detection using the method of the first aspect of this disclosure. Furthermore, since the purpose of the reaction is to add an RNA polymerase promoter sequence to a target nucleic acid present, and it is not necessary to measure the amplification product, the number of cycles of the amplification reaction need not be as high as the 35-40 cycles typically performed in amplification reactions.

[0055] In one embodiment, an RNA polymerase promoter sequence is added to a target sequence by an amplification reaction, the amplification reaction comprising: - Providing a sample of genetic material; - denaturing said sample of genetic material; - adding at least one target primer pair under conditions that allow annealing of said primers to said sample of genetic material, The target primer pair comprises: - a sequence specific for said target sequence; and - RNA polymerase promoter sequence; a first primer comprising: - a sequence specific for said target sequence; a second primer comprising: Including, the sequences in the primers specific for the target sequence are selected to allow amplification of the target nucleic acid sequence if the target nucleic acid sequence is present in the sample of genetic material; - performing a predetermined number of cycles of an amplification reaction to amplify the target nucleic acid sequence present in said sample of genetic material and to add a promoter sequence for RNA polymerase; This includes a series of steps.

[0056] As used herein, the term "primer" refers to an oligonucleotide-length nucleic acid that, when duplexed with a polynucleotide template, can serve as an initiation point for nucleic acid synthesis and can be extended along the template from its 3' end, thereby forming an extended duplex (duplex). As used herein, the term "primer pair" refers to at least two primers, one referred to as the "forward primer" and the other referred to as the "reverse primer," that are complementary to nucleotide sequences that flank, i.e., at the beginning and end, respectively, of a fragment of a template nucleic acid to be amplified.

[0057] As one of skill in the art will readily appreciate, the amplification reaction in this embodiment utilizes at least one primer pair that flanks the target region of interest, as shown generally in FIG.

[0058] The amplification reaction of this embodiment may be adapted for various subsequent aspects of detecting the presence of the target (also referred to as the "detection step").

[0059] Thus, in embodiments where the detection step is designed to detect the presence of a plurality of different target nucleic acid sequences, the amplification reaction prior to the detection step is suitably designed to include a plurality of different primer pairs, each primer pair adapted to amplify each target nucleic acid sequence to be detected. In this embodiment of the amplification reaction, the addition of at least one pair of target primers includes the addition of a plurality of pairs of target primers, each primer pair including a sequence specific for a different target sequence, such that the polymerase chain reaction amplifies all the different target sequences present in the genetic material sample.

[0060] In some aspects, the amplification reaction is also used to add a sequence to the target nucleic acid that enables the target nucleic acid to bind, directly or indirectly, to a capture nucleic acid disposed on a solid support.

[0061] In embodiments of the detection step in which the or each capture nucleic acid is identical or complementary to at least a portion of the target nucleic acid sequence and binds directly to one of the strands of a double-stranded nucleic acid to be detected, the or each second primer in the amplification reaction comprises a sequence that is identical or complementary to at least a portion of the sequence of the or each capture nucleic acid. Amplification using such a second primer incorporates a sequence in the amplified target nucleic acid that hybridizes to the capture nucleic acid when the amplified target nucleic acid is introduced into the reaction chamber in single-stranded form for detection.

[0062] In one embodiment, the second primer may contain a sequence identical or complementary to the target sequence, adding such target-specific nucleic acid sequence to the amplified target nucleic acid. In this embodiment of the detection step, each capture nucleic acid is designed to have a nucleic acid sequence that is identical or complementary to at least a portion of the target-specific nucleic acid sequence, allowing the amplified target nucleic acid to hybridize with the capture nucleic acid.

[0063] Alternatively, the second primer may contain a nucleic acid sequence that is not specific to the target in addition to the sequence that is identical or complementary to the target sequence, and such a non-target specific nucleic acid sequence is also added to the amplified target nucleic acid. In this embodiment of the detection step, each capture nucleic acid is designed to have a nucleic acid sequence that is identical or complementary to at least a portion of the non-target specific nucleic acid sequence, allowing the amplified target nucleic acid to hybridize with the capture nucleic acid.

[0064] In an alternative embodiment, based on the more flexible concept of "capture-adapter-target" discussed in the detection step above, the or each second primer is designed accordingly, and an amplification reaction is instead used to introduce a sequence complementary to the adapter sequence into the target nucleic acid. In this embodiment, the or each second primer then further comprises a specific adapter sequence, while the or each capture nucleic acid comprises a unique adapter sequence. The reaction chamber further comprises at least one adapter nucleic acid comprising a first specific adapter sequence identical to the specific adapter sequence in the or each second primer, and a second unique adapter sequence complementary to the unique adapter sequence of the capture nucleic acid, and the or each capture nucleic acid indirectly binds to the corresponding target nucleic acid by overlapping hybridization of the or each adapter nucleic acid with both the capture nucleic acid and the or each second primer. Thus, advantageously, in embodiments where multiple target sequences are interrogated, different adapter sequences can then be added to different target nucleic acids, such that each target nucleic acid to be detected contains its own adapter sequence that is useful for binding only to adapter nucleic acids that contain that specific adapter sequence, and every adapter nucleic acid also contains a unique adapter sequence that is complementary only to a unique sequence on the corresponding capture nucleic acid.

[0065] In a more specific embodiment of this advantageous setting, further advantages may be obtained by designing the adapter nucleic acid to include additional target sequences adjacent to the first specific adapter sequence (i.e. adjacent to a stretch of sequence also present in the or each second primer). Beyond the portion of the target sequence provided by the or each second primer, there is an additional sequence match between the amplified target sequence and the adapter sequence, so that the complementary range of the target nucleic acid and the adapter nucleic acid is increased when the target nucleic acid is indirectly bound to the capture nucleic acid. A further advantage of such a design is that the annealing between the target nucleic acid and the adapter nucleic acid can then be performed at a temperature higher than the temperature required for primer hybridization. As a result, the interference of excess primers in the hybridization between the target nucleic acid and the adapter nucleic acid is suppressed or eliminated. Furthermore, the presence of a target portion in the adapter nucleic acid that is slightly longer than the target sequence in the or each second primer also provides additional selectivity against undesired by-products (e.g. primer-dimers) when extension conditions are applied to synthesize a complementary strand of the single-stranded target nucleic acid in the detection step (Figure 1, step (2)).

[0066] In one embodiment, the amplification reaction is a polymerase chain reaction (PCR).

[0067] Performing an amplification reaction (e.g. PCR) on a sample of genetic material in preparation of a target nucleic acid for subsequent detection has the advantage that additional sequences designed to bind directly or indirectly to the RNA polymerase promoter sequence or capture nucleic acid can be added to the target nucleic acid by introducing these sequence elements into the or each primer pair used to amplify one or more specific desired target sequences in the genetic material sample. Another advantage of amplifying (e.g. PCR) before detection in this manner is that reagents in the amplification reaction mixture, such as DNA polymerase and dNTPs, are potential useful components in the extension step of the detection stage. In this embodiment, the reaction mixture from the amplification reaction may simply be transferred to the reaction chamber of the detection stage without further manipulation and used directly. However, this embodiment of the method also benefits from the fact that amplification and detection are separated into separate reaction steps and are carried out sequentially under different reaction conditions. Thus, the amplification reaction does not need to continue long enough to generate a detectable number of protons by itself, but only long enough to amplify a sufficient amount of the target nucleic acid with the added RNA polymerase promoter sequence. This means that the analysis can be carried out quickly and efficiently. Similarly, detection of protons using, for example, a temperature-sensitive ISFET is not exposed to the high temperatures and temperature changes associated with thermal cycling in amplification methods such as PCR, resulting in a longer lifetime and greater reliability of the instruments and detection units, thus avoiding a major drawback of conventional pH sensor detection methods.

[0068] In some embodiments where amplification is performed to add required sequences (i.e., promoter sequences for RNA polymerase and additional sequences for direct or indirect binding to the capture nucleic acid), the predetermined number of cycles of the amplification reaction may be only one cycle, which serves to add the sequence to the target nucleic acid present in the genetic material sample. In some embodiments, the predetermined number of cycles is 1 to 40 cycles, e.g., 1 to 30 cycles, 1 to 20 cycles, 1 to 10 cycles, 2 to 10 cycles. In specific embodiments, the predetermined number of cycles is 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles.

[0069] The methods described herein can be automated. Thus, in a preferred embodiment of this disclosure, the disclosed methods are performed by an automated system. In this specification, the term "automated system" may refer to an integrated platform that includes equipment and disposable materials, such as plastics and solutions, that the system uses in an automated manner to complete certain steps. Such steps can be initiated by a user, and no user intervention is required during the automated processing of the system until the steps are completed. In this specification, the term "equipment" should be understood as a machine that includes at least a user interface (e.g., at least a start button or an electrical plug), a computer with software programmed to perform functions such as assaying according to the methods disclosed herein. This may include, for example, mixing, heating, data detection, data collection, data analysis, etc. In a preferred embodiment, the disposable materials are provided in the form of a kit. In this specification, the term "kit" should be interpreted as a set that includes at least one item, or an assembly of items or devices required for a specific purpose, such as performing a molecular biology method or assay. A second aspect of this disclosure provides a kit comprising a reaction chamber having a detection zone for detecting proton release / accumulation during transcription of a nucleic acid, a capture nucleic acid disposed on a solid support and adapted to indirectly or directly bind to a target nucleic acid, and reagents for applying conditions for nucleic acid extension and transcription.

[0070] Also provided herein is the use of the methods and articles of manufacture (such as kits, automated systems, etc.) described herein for detecting at least one target nucleic acid sequence in a sample, which comprises adding an RNA polymerase sequence to the target nucleic acid and detecting transcription activity, e.g., protons released by the activity of the RNA polymerase.

[0071] A third aspect of this disclosure provides various in vitro methods for using the presence, absence or amount of a target nucleic acid in a sample as the basis for a subject's clinical decision.

[0072] In certain embodiments, the method is an in vitro diagnostic method that includes a further step in which the presence, absence or amount of at least one target nucleic acid in a sample is used as a basis for diagnosing a condition in a subject.

[0073] In another embodiment, the method is an in vitro prognostic method that includes a further step of using the presence, absence or amount of at least one target nucleic acid in the sample as a basis for determining the prognosis of the subject's condition.

[0074] In yet another aspect, the method is an in vitro subject stratification method that includes a further step of using the presence, absence or amount of at least one target nucleic acid in a sample as a basis for predicting the likelihood of successful treatment of a subject's condition.

[0075] In a related aspect, the method is an in vitro subject stratification method that includes a further step of using the presence, absence or amount of at least one target nucleic acid in a sample as a basis for predicting the likelihood of resistance to treatment of a condition in a subject.

[0076] In another aspect, the method is an in vitro method for selecting an appropriate treatment for a subject's condition, comprising a further step of using the presence, absence or amount of at least one target nucleic acid in the sample as a basis for selecting an appropriate treatment for the subject's condition.

[0077] A preferred embodiment of such a method comprises: - obtaining a sample of genetic material from the subject to be tested or providing a previously obtained sample of genetic material; - concentrating the target nucleic acid present in said sample of genetic material to enable detection by the methods disclosed herein; may also include

[0078] In certain embodiments of such methods, detection is achieved by adding an RNA transcription promoter sequence via an amplification reaction, and enrichment is achieved by an amplification reaction using primers designed to amplify a target nucleic acid from a sample of genetic material.

[0079] In certain aspects of such methods, the sample of genetic material is obtained from a sample taken from a subject. As previously defined, the sample may be selected from the group consisting of cell cultures, body fluids, body fluid sediments, lavage samples, fine needle aspirates, biopsy samples, tissue samples, cancer cells, other cells obtained from a subject, cells derived from tissues of a subject undergoing testing and / or treatment for a disease or infection or cells cultured in vitro, and forensic samples. If the sample is a body fluid, it may be selected from the group consisting of whole blood, bone marrow, cerebrospinal fluid (CSF), peritoneal fluid, pleural fluid, lymph, serum, plasma, urine, chyle, faeces, sperm, sputum, nipple aspirate, saliva, swab samples, wash / lavage fluid and brush samples.

[0080] In certain aspects of such methods, the subject is a mammal, hi certain aspects, the subject is a human.

[0081] Also provided are kits for diagnosing, determining a prognosis, stratifying a subject for treatment, and / or selecting an appropriate therapy for a subject. Such kits may incorporate any of the various features, aspects and embodiments mentioned in connection with the various methods and uses disclosed.

[0082] Once the general principles disclosed herein are provided to those skilled in the art of modern biochemistry and genetic technology, it is believed to be within the skill of such a person to design and embody the various nucleic acid elements for use in the various aspects of this disclosure. As an example, the skilled person can select a suitable target sequence to detect in a genetic material sample, including the length of the target-specific sequence in the primer and other design considerations, and an associated primer pair for the detection and amplification of such a target sequence. Similarly, the skilled person can design a suitable capture nucleic acid sequence to be immobilized on a solid support, and, if applicable, a suitable adapter nucleic acid sequence having the required degree of overlap with the target nucleic acid sequence and the capture nucleic acid sequence, respectively. Furthermore, in aspects in which amplification is performed to add the required sequence element to the target nucleic acid, it is within the ability of the skilled person to select the appropriate reaction parameters for such amplification, such as the reaction temperature and components of the reaction mixture, including the nature and concentrations of the polymerase enzyme, dNTPs or NTPs, and other known factors.

[0083] Also, while the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope of the disclosure of the specification. In addition, many modifications can be made to adapt a particular situation to the teachings of the present invention without departing from the essential scope of the invention. Therefore, it is intended that the present invention not be limited to any particular embodiment contemplated, but rather to include all embodiments falling within the scope of the appended claims. [Brief description of the drawings]

[0084] [Figure 1] FIG. 1 is a schematic diagram of one embodiment of the method of this disclosure, in which capture nucleic acids are immobilized at two array addresses i and j on a solid support in a reaction chamber, and target nucleic acids present in a sample are directly bound to the capture nucleic acids. [Diagram 2]FIG. 2 is a schematic diagram of another embodiment of the method of this disclosure, in which capture nucleic acids are immobilized at two array addresses i and j of a solid support in a reaction chamber, and target nucleic acids present in a sample are indirectly bound to the capture nucleic acids via adapter nucleic acids. [Diagram 3] FIG. 3 is a schematic diagram of a primer design useful in one embodiment of the disclosed method in which amplification is performed to add a promoter sequence for an RNA polymerase to a target nucleic acid to be detected. [Figure 4] FIG. 4 shows the current (y-axis) measured in reactions of various concentrations (x-axis) of template target nucleic acid in the experiment described in Example 1. [Diagram 5] FIG. 5 is a photograph of an ISFET sensor array with capture nucleic acids immobilized using click chemistry as described in Example 2. [Figure 6] FIG. 6 is a graph of output current (y-axis) versus time (x-axis) in the presence (A) or absence (B) of target sequence after baseline subtraction for the four sensors shown in the inset of FIG. 6A and described in Example 2. EXAMPLES

[0085] The following examples illustrate aspects of this disclosure implemented in a variety of settings.

[0086] Example 1 An array of ISFET sensors (Taiwan Semiconductor Manufacturing Company; TSMC) was used to detect pH changes resulting from T7 RNA polymerase activity of a model target nucleic acid containing a T7 promoter sequence. The size of the array was 850 × 850 μm. 2The array contained 1024 sensors with hafnium oxide (HfO2) as the pH-sensitive layer. The sensors were arranged in 32 rows and 32 columns. The array was fabricated as a standard complementary metal oxide semiconductor (CMOS) and placed on a printed circuit board that provided electrical connections to the measurement station (called Demobox) and interfaced with a laptop running control software provided by TSMC. A polymer confinement well was used to confine the buffer solution to a sensing area with a volume of 20 μL. The buffer solution was biased using a leak-free miniature Ag / AgCl reference electrode (eDAQ) connected to the Demobox.

[0087] As the target nucleic acid, pSP73 DNA plasmid, which is known to contain the promoter sequence of T7 RNA polymerase upstream of the multiple cloning site, was used in the experiment. A mixture of the plasmid, CutSmart® 10x buffer and the restriction enzyme HPAI in RNAse-free water was heated at 37°C for 1 hour to cut and linearize the plasmid at a specific sequence. All reagents were obtained from Integrated DNA Technologies (IDT). Subsequently, a master mix of T7 RNA polymerase was prepared in CHES buffer with a final composition of 20 mM NaCl, 6 mM MgCl2, 10 Mm DTT, 1 Mm spermidine, 12 U / μL T7 enzyme and 0.5 mM of each NTP. The pH was adjusted to about 8 with NaOH. Prior to the experiment, the pH sensitivity of the ISFET sensor array chip was tested in 1 M CHES buffer for pH 7.5-8.5 in increments of 0.2.

[0088] Target nucleic acids prepared as described previously were added at various concentrations to a master mix of T7 RNA polymerase to a final volume of 20 μL and quickly pipetted into the confinement wells attached to the chip of an ISFET sensor array connected to a Demobox.

[0089] The software was programmed to measure the output current from every sensor in the array every second. Multiple experiments were performed in the same manner for various concentrations of target nucleic acid. T7 RNA polymerase buffer alone was used as a negative control.

[0090] The results are shown in Figure 4. This figure shows the absolute output current measured 2 minutes after the start of the experiment (i.e., the moment the buffer solution was placed on the chip) versus the logarithm of the target concentration on a representative sensor. The measured current changes with pH, ​​since different target concentrations result in different pH changes. Specifically, since the ISFET is an n-channel transistor, the current is expected to decrease with decreasing target concentration (reduced proton production). The lowest concentration of linearized plasmid tested was 10 -6 ng / μL (approximately 10 4 molecule) was also recognized by the sensor at this concentration when compared to the negative control.

[0091] Example 2 A proof-of-concept system was tested and designed using three oligonucleotides acting as capture probes, adapters and target nucleic acids. The oligonucleotides were dissolved in IDTE buffer (pH 8) to 100 μM.

[0092] [Table 1]

[0093] To test the setup, four different capture probes (Table 1; SEQ ID NOs: 3-6) were immobilized on an array of ISFET sensors by click chemistry (Movilli et al (2020), ACS Langmuir 36:4272-4279). First, the chip obtained from TSMC was wet and dry cleaned with solvent and mild ozone. It was then functionalized by click chemistry using 3-azidopropyltriethoxysilane (Gelest) at 70 °C for 6 h to form azide groups on the surface that can react efficiently with DBCO-modified oligonucleotides. DBCO-functionalized capture probes were designed and spotted on the azide-functionalized chips with sciFLEXARRAYER SX (Scienion) at a concentration of 10 μM in 1 nL of buffer (1 M NaCl and 10 mM Tris HCl (pH 8)). The spotted chip was incubated in a high humidity environment (85%) to avoid evaporation, and the click chemistry reaction was allowed to proceed for 1 h. After 1 h, the chip was rinsed with deionized water. A photograph of the array spotted with capture probes is shown in Figure 5.

[0094] A test to confirm the success of the click chemistry reaction was performed as follows. Four different test probes (Table 1; SEQ ID NOs: 7-10) carrying fluorescent molecules and complementary to the spotted capture probes were hybridized on the spotted chip at room temperature for 1 hour. Then, the fluorescence was observed under an optical microscope equipped with excitation / emission filters. It was established that the optical signal originated from the spotted area, which means that the hybridization of the test probes on each spotted capture probe was successful.

[0095] [Table 2]

[0096] After successful testing, the adapter (SEQ ID NO: 11) and target (SEQ ID NO: 12) nucleic acid molecules were hybridized with the capture probe (SEQ ID NO: 13) spotted on the chip (Table 2). The sequences of the adapter and target oligonucleotides were designed such that when hybridized, they were double-stranded, with the promoter sequence of T7 RNA polymerase on the side exposed to the buffer. The adapter and target oligonucleotides could only hybridize on specific spots on the array due to their complementarity with the capture probe at that position, and no hybridization occurred on spots with no capture probe or with a non-complementary capture probe. A solution of 500 nM each of the two oligonucleotides was prepared in 1 M NaCl, 10 mM Tris HCl buffer, transferred directly to the ISFET sensor array using a pipette, and left at room temperature for 1 hour. After 1 hour, the chip was washed with the same solution. At this point, the ISFET sensor chip was ready to be tested using the setup described in Example 1. The chip was inserted into a Demobox that was fitted with a buffer containment well and programmed to measure the output from all sensors in the array over time. A master mix of T7 RNA polymerase of the same composition as described in Example 1 was used.

[0097] Figure 6 shows the output current from each sensor, reflecting the spot pattern. The chemical composition of the spotted and non-spotted locations is different, with the areas outside the spots only having a silanized layer with azide groups, and the spots having oligonucleotides on top of this layer, and therefore the electrochemical potential also results in a current value.

[0098] Figures 6A and 6B show graphs of current versus time after baseline subtraction for the four sensors highlighted in the inset. The sensors and curve sets are coded as indicated in the legend. First, an enzyme-free T7 RNA polymerase master mix was placed on the chip to stabilize the electrical output signal. In this condition, no reaction was initiated due to the absence of enzyme. This enzyme-free buffer was then pipetted out of the wells and replaced with a buffer with all the components required to initiate the reaction.

[0099] As shown in Figure 6A, the pixel with hybridized target nucleic acid (containing the T7 promoter sequence) (top left corner, pixel 7.7) has a faster transient response compared to the pixel lacking the target upon introduction of the master mix of T7 RNA polymerase with the enzyme. The reason for this faster response is likely because the reaction is initiated on the sensor with the hybridized target containing the promoter sequence of T7 RNA polymerase, so that the protons generated upon nucleotide insertion are immediately detected by the ISFET. However, eventually, a response is obtained from the distant sensor by diffusion across the chip.

[0100] As a negative control, a time response was also obtained from a chip in which capture probes were similarly spotted but on which no target nucleic acid was hybridized. The current vs. time response of this chip after baseline subtraction is shown in Figure 6B. No delay was observed between the responses of pixels at different locations, which is due to the fact that no reaction occurs and all sensors are exposed to the same chemical buffer composition (electrochemical potential) simultaneously.

[0101] List of embodiments by item 1. A method for detecting the presence of at least one target nucleic acid sequence in a sample, comprising: - providing a sample suspected of containing said at least one target nucleic acid sequence; - adding a promoter sequence for an RNA polymerase to a target nucleic acid sequence present in said sample; - said sample - at least one detection zone; and - at least one capture nucleic acid disposed on a solid support and adapted to indirectly or directly bind to said target nucleic acid; into a reaction chamber having generating a sequence of single-stranded nucleic acid bound directly or indirectly to the capture nucleic acid disposed on the solid support; - applying extension conditions that allow the generation of a nucleic acid strand complementary to said single-stranded nucleic acid to form a double-stranded nucleic acid that contains a promoter sequence for an RNA polymerase and is directly or indirectly bound to said capture nucleic acid disposed on said solid support; - applying transcription conditions that allow the production of transcripts from the double-stranded nucleic acid captured on the solid support, the production of transcripts releasing protons as transcription proceeds; and - detecting the presence of said protons as a signal from said detection zone, said signal being indicative of the presence of said target nucleic acid sequence in said sample; A method comprising a series of steps. 2. The method of claim 1 for detecting the presence of multiple target nucleic acid sequences, wherein the multiple target sequences correspond to multiple capture nucleic acids arranged on a solid support, each adapted to indirectly or directly bind to a different target nucleic acid sequence. 3. The method of claim 2, wherein the plurality of capture nucleic acids are arranged in the form of an array on the solid support, each capture nucleic acid representing an addressable location on the array. 4. The method of claim 3, wherein the detection signal from the detection zone is identified as arising from a specific addressable location on the array. 5. A method according to any of the preceding claims, wherein at least a portion of the sequence of the or each capture nucleic acid is identical to or complementary to at least a portion of the target nucleic acid sequence and binds directly to one of the strands of the double-stranded nucleic acid to be detected. 6 - the step of adding a promoter sequence for an RNA polymerase further comprises the step of adding a specific adapter sequence to a target nucleic acid sequence present in the sample; - the or each capture nucleic acid comprises a unique adapter sequence; and - the reaction chamber comprises: - a first specific adapter sequence that is identical or complementary to the specific adapter sequence in the target nucleic acid sequence present in the sample; and - a second unique adapter sequence that is complementary to the unique adapter sequence of the capture nucleic acid; and further comprising at least one adaptor nucleic acid comprising the or each capture nucleic acid indirectly binds to the corresponding target nucleic acid to be detected by overlapping hybridization of the or each adaptor nucleic acid with both the capture nucleic acid and the or each target nucleic acid; The method according to any one of Items 1 to 4. 7. The method according to any of the preceding claims, wherein the steps of providing the sample and adding a promoter sequence for an RNA polymerase and, if present, a specific adapter sequence, are carried out as part of an amplification reaction. 8 The amplification reaction is - Providing a sample of genetic material; - denaturing said sample of genetic material; - adding at least one target primer pair under conditions that allow annealing of said primers to said sample of genetic material, The target primer pair comprises: - a sequence specific for said target sequence; and - RNA polymerase promoter sequence; a first primer comprising: - a sequence specific for said target sequence; a second primer comprising: Including, the sequences in the primers specific for the target sequence are selected to allow amplification of the target nucleic acid sequence if the target nucleic acid sequence is present in the sample of genetic material; - performing a predetermined number of cycles of an amplification reaction to amplify the target nucleic acid sequence present in said sample of genetic material and to add a promoter sequence for RNA polymerase; Item 8. The method according to Item 7, comprising the steps of: 9. The method of claim 8, wherein adding at least one target primer pair comprises adding a plurality of target primer pairs, each primer pair comprising a sequence specific for a different target sequence, and wherein the amplification reaction amplifies all different target sequences present in the sample of genetic material. 10. The method according to any one of items 7 to 9, wherein at least a portion of the sequence of the or each capture nucleic acid is identical to or complementary to the first primer or each second primer and directly binds to one of the strands of the or each target nucleic acid.

[0102] 11 - the or each second primer further comprises a specific adapter sequence; - the or each complementary nucleic acid comprises a unique adapter sequence; and - the reaction chamber comprises: - a first specific adapter sequence that is identical to the specific adapter sequence in the or each second primer; and - a second unique adapter sequence that is complementary to the unique adapter sequence of the complementary nucleic acid; and further comprising at least one adaptor nucleic acid comprising 10. The method according to any one of items 7 to 9, wherein the or each capture nucleic acid indirectly binds to the corresponding target nucleic acid by overlap hybridization between the or each adaptor nucleic acid and both the capture nucleic acid and the or each second primer. 12. The method of claim 11, wherein the at least one adaptor nucleic acid further comprises an additional target sequence adjacent to the first specific adaptor sequence, increasing the extent of complementarity between the desired amplicon and the adaptor nucleic acid beyond the portion of the target sequence provided by the second primer. 13. The method according to any one of items 7 to 12, wherein the amplification reaction is a polymerase chain reaction (PCR). 14. The method according to any of the preceding claims, wherein the detection zone comprises a detection unit, such as an ion-sensitive field effect transistor. 15. The method according to any of the preceding paragraphs, wherein the extension conditions include a reaction temperature of 75 to 90°C. 16. The method of any of the preceding claims, wherein the extension conditions comprise the presence of a DNA polymerase. 17. The method according to any of the preceding paragraphs, wherein the transcription conditions include a reaction temperature of 20 to 40°C. 18. The method of any of the preceding claims, wherein the transcription conditions include the presence of an RNA polymerase. 19. The method according to any of the preceding claims, wherein the promoter sequence of the RNA polymerase is a promoter sequence of T7 RNA polymerase. 20. The method of claim 18 or 19, wherein the RNA polymerase is T7 RNA polymerase.

[0103] 21. The method of any of the preceding claims which is an in vitro method of diagnosis, prognosis, stratification of a patient condition, or selection of a therapy, comprising the additional step of using the presence, absence, or amount of at least one target nucleic acid in the sample as a basis for diagnosing a subject's condition, prognosis of a subject's condition, stratification of a patient, or selection of a therapy for a patient. 22 - Obtaining a sample of genetic material from the subject to be tested; - concentrating the target nucleic acid present in said sample of genetic material to enable its detection by the method according to any one of claims 1 to 18; 22. The method according to claim 21, comprising: 23. The method according to claim 22, wherein the detection is carried out by the method according to any one of claims 7 to 13, and the enrichment is carried out by an amplification reaction using primers designed to amplify the target nucleic acid from the sample of genetic material. 24. The method according to any one of claims 21 to 23, wherein the sample of genetic material is obtained from a sample taken from the subject. 25. The method of claim 24, wherein the sample is selected from the group consisting of cell cultures, body fluids, body fluid sediments, lavage specimens, fine needle aspirates, biopsy samples, tissue samples, cancer cells, other cells obtained from a subject, cells derived from tissue or in vitro cultured cells of a subject being tested and / or treated for disease or infection, and forensic samples. 26. The method of claim 25, wherein the sample is a body fluid selected from the group consisting of whole blood, bone marrow, cerebrospinal fluid (CSF), peritoneal fluid, pleural fluid, lymphatic fluid, serum, plasma, urine, chyle, stool, sperm, sputum, nipple aspirate, saliva, swab specimen, wash / lavage fluid and brush specimen. 27. The method according to any one of items 21 to 26, wherein the subject is a mammal, for example a human.

Claims

1. 1. A method for detecting the presence of at least one target nucleic acid sequence in a sample, comprising: - providing a sample suspected of containing said at least one target nucleic acid sequence; - adding a promoter sequence for an RNA polymerase to a target nucleic acid sequence present in said sample; - the sample at least one detection zone; and at least one capture nucleic acid arranged on a solid support and adapted to bind indirectly or directly to said target nucleic acid; into a reaction chamber having generating a sequence of single-stranded nucleic acid bound directly or indirectly to a capture nucleic acid disposed on a solid support; - applying extension conditions that allow the generation of a nucleic acid strand complementary to the single-stranded nucleic acid to form a double-stranded nucleic acid that contains a promoter sequence for an RNA polymerase and is directly or indirectly bound to the capture nucleic acid disposed on the solid support; - applying transcription conditions that allow the production of transcripts from the double-stranded nucleic acid captured on the solid support, the production of transcripts releasing protons as transcription proceeds; and - detecting the presence of said protons as a signal from said detection zone, said signal being indicative of the presence of said target nucleic acid sequence in said sample; A method comprising the following series of steps.

2. 10. The method of claim 1 for detecting the presence of multiple target nucleic acid sequences, wherein the multiple target sequences correspond to multiple capture nucleic acids disposed on a solid support, each adapted to indirectly or directly bind to a different target nucleic acid sequence.

3. 3. The method of claim 2, wherein the plurality of capture nucleic acids are arranged in the form of an array on the solid support, each capture nucleic acid representing an addressable location on the array.

4. 2. The method of claim 1, wherein at least a portion of the sequence of the or each capture nucleic acid is identical to or complementary to at least a portion of the target nucleic acid sequence and binds directly to one of the strands of the double-stranded nucleic acid to be detected.

5. - the step of adding a promoter sequence for an RNA polymerase further comprises adding a specific adapter sequence to a target nucleic acid sequence present in the sample; - the or each capture nucleic acid comprises a unique adapter sequence; and said reaction chamber comprising: - a first specific adapter sequence that is identical to or complementary to the specific adapter sequence in the target nucleic acid sequence present in the sample; and - a second unique adapter sequence that is complementary to the unique adapter sequence of the capture nucleic acid; and further comprising at least one adaptor nucleic acid comprising: the or each capture nucleic acid indirectly binds to the corresponding target nucleic acid to be detected by overlapping hybridization of the or each adapter nucleic acid with both the capture nucleic acid and the or each target nucleic acid; The method of claim 1.

6. 2. The method of claim 1, wherein the steps of providing the sample and adding a promoter sequence for an RNA polymerase and, if present, a specific adapter sequence, are performed as part of an amplification reaction.

7. The amplification reaction - providing a sample of genetic material; - denaturing said sample of genetic material; - adding at least one target primer pair under conditions that allow annealing of said primers to said sample of genetic material, The target primer pair a sequence specific for said target sequence; and - RNA polymerase promoter sequence; a first primer comprising: a sequence specific for said target sequence; a second primer comprising: Including, the sequences in the primers specific for the target nucleic acid sequence are selected to allow amplification of the target nucleic acid sequence if the target nucleic acid sequence is present in the sample of genetic material; - carrying out a predetermined number of cycles of an amplification reaction to amplify the target nucleic acid sequence present in said sample of genetic material and to add a promoter sequence for RNA polymerase; The method according to claim 6, comprising the steps of:

8. 8. The method of claim 7, wherein adding at least one target primer pair comprises adding multiple target primer pairs, each primer pair comprising a sequence specific for a different target sequence, and wherein the amplification reaction amplifies all different target sequences present in the sample of genetic material.

9. 7. The method of claim 6, wherein at least a portion of the sequence of the or each capture nucleic acid is identical to or complementary to the or each first primer and binds directly to one of the strands of the or each target nucleic acid.

10. - the or each second primer further comprises a specific adapter sequence; - the or each complementary nucleic acid comprises a unique adapter sequence; and said reaction chamber comprising: - a first specific adapter sequence that is identical to the specific adapter sequence in the or each second primer; and - a second unique adapter sequence that is complementary to the unique adapter sequence of the complementary nucleic acid; and further comprising at least one adaptor nucleic acid comprising:

7. The method of claim 6, wherein the or each capture nucleic acid indirectly binds to the corresponding target nucleic acid by overlapping hybridization of the or each adapter nucleic acid with both the capture nucleic acid and the or each second primer.

11. 11. The method of claim 10, wherein the at least one adapter nucleic acid further comprises an additional target sequence adjacent to the first specific adapter sequence, increasing the extent of complementarity between the desired amplicon and the adapter nucleic acid beyond the portion of the target sequence provided by the second primer.

12. The method of claim 6, wherein the amplification reaction is a polymerase chain reaction (PCR).

13. The method of claim 1 , wherein the detection zone comprises a detection unit, such as an ion-sensitive field effect transistor.

14. The method of claim 1 , wherein the extension conditions comprise the presence of a DNA polymerase.

15. The method of claim 1 , wherein the transcription conditions include the presence of an RNA polymerase.

16. The method of claim 1, wherein the promoter sequence of the RNA polymerase is a promoter sequence of T7 RNA polymerase.

17. 10. The method of claim 1, which is an in vitro method of diagnosis, prognosis, stratification of a subject's condition, or selection of a therapy, comprising the additional step of using the presence, absence, or amount of at least one target nucleic acid in the sample as a basis for diagnosing a subject's condition, prognosis of a subject's condition, stratification of the patient, or selection of a therapy for the patient.

18. - obtaining a sample of genetic material from the subject to be tested; - concentrating the target nucleic acids present in said sample of genetic material to enable their detection by the method of claim 1; 18. The method of claim 17, comprising:

19. 19. The method of claim 18, wherein the detection is performed by the method of claim 6 and the enrichment is performed by the amplification reaction using primers designed to amplify the target nucleic acid from the sample of genetic material.

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