Ferrocene labeled primers for electrochemical detection

Abstract

The present invention provides novel primers, wherein a ferrocene marker is attached to the primers. The ferrocene label is incorporated into the amplification product. When the amplification product incorporating the ferrocene label is denatured, it is capable of binding to a capture probe and of detecting the presence or absence of the ferrocene label via electrochemical detection. The system avoids the use of signal probes in sandwich assays that have been used during electrochemical detection.

Description

Technical Field

[0001] The present invention relates generally to the fields of electrochemical detection techniques and molecular diagnostics. Background Art

[0002] Access to diagnostic tests is critical to helping limit the spread of disease. However, access to effective diagnostic tests has been a significant issue during the COVID-19 pandemic, especially during the initial outbreak in 2020. Accelerating the development of COVID-19 tests and scaling up their manufacturing and delivery are critical in the fight against COVID-19. There remains a need to rapidly develop accurate, rapid assays and manufacture these assays so that they can be brought to market quickly as additional COVID-19 variants and new infectious diseases emerge. Summary of the invention

[0003] Provided herein are methods, devices and systems for amplifying and detecting nucleic acids (such as DNA and / or RNA) using signal primers used in electrochemical detection systems. Using signal primers to amplify and detect nucleic acids (such as DNA and / or RNA) can accelerate the development of detection assays, improve their accuracy, speed up the time to obtain results and simplify manufacturing to ensure that diagnostic kits can be quickly developed and delivered to patients in need. Rapid development and distribution of diagnostic kits will help reduce the spread of disease.

[0004] In a first aspect, a method for detecting the presence or absence of a target nucleic acid in a sample is provided, the method comprising: (a) combining a solution containing or suspected of containing a target nucleic acid with an amplification reagent to amplify the target nucleic acid in the presence of the target nucleic acid, the amplification reagent comprising a labeled primer capable of hybridizing with the target nucleic acid; (b) amplifying the target nucleic acid in the presence of the target nucleic acid to produce a double-stranded labeled amplicon; (c) incubating the double-stranded labeled amplicon with an exonuclease to form a single-stranded labeled amplicon; (d) hybridizing the single-stranded labeled amplicon with a first capture probe; and (e) using electrochemical detection to detect the presence or absence of the target nucleic acid in the sample. In some embodiments, the labeled primer, the double-stranded labeled amplicon, and / or the single-stranded labeled amplicon comprises at least one label selected from the group consisting of ferrocene, methylene blue, or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the labeled primer comprises a ferrocene label at the 5'-end, at any one of nucleotides 1 to 3 at the 5'-end, at the 3'-end, at any one of nucleotides 1 to 3 at the 3'-end, or at both the 5'-end and the 3'-end. In some embodiments, the labeled primer comprises a ferrocene label inside the labeled primer. In some embodiments, the labeled primer, the double-stranded labeled amplicon and / or the single-stranded labeled amplicon comprise multiple ferrocene labels at at least two positions. In some embodiments, the single-stranded labeled amplicon comprises a first segment capable of hybridizing with the first capture probe, a second segment that cannot be bound to the capture probe, and a third segment comprising the at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue, or osmium. In some embodiments, the labeled primer comprises a first segment capable of hybridizing with the first capture probe, a second segment comprising a joint, and a third segment comprising the at least one label, wherein the joint connects the first segment and the third segment. In some embodiments, the method further comprises: separating the labeled primer and the single-stranded labeled amplicon before electrochemical detection. In another embodiment, the method further comprises: separating the labeled primer and the double-stranded labeled amplicon before electrochemical detection. In some embodiments, the labeled primer consists of a first segment capable of hybridizing with the capture probe and a second segment comprising the at least first label. In some embodiments, the amplification reagent comprises a nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer necessary for the function of the nucleic acid polymerase, and a divalent cation (such as, for example, Mg). 2+ and / or Mn 2+). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises the step of using a reverse transcriptase to reverse transcribe the RNA to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity.

[0005] In a second aspect, a method for detecting the presence or absence of a target nucleic acid in a sample, the method comprising: (a) receiving a sample; (b) extracting nucleic acid from the sample, wherein the nucleic acid is suspected of containing a target nucleic acid; (c) combining the nucleic acid with an amplification reagent to amplify the target nucleic acid in the presence of the target nucleic acid, the amplification reagent comprising a signal primer capable of hybridizing with the target nucleic acid; (d) amplifying the target nucleic acid in the presence of the target nucleic acid to produce a double-stranded signal amplifier; (e) incubating the double-stranded signal amplifier with an exonuclease to form a single-stranded signal amplifier; (f) hybridizing the single-stranded signal amplifier with a first capture probe; and (g) using electrochemical detection to detect the presence or absence of the target nucleic acid in the sample. In some embodiments, the signal primer comprises an electrochemically detectable label. In some embodiments, the signal primer comprises at least one label selected from the group consisting of ferrocene, methylene blue or osmium. In certain embodiments, the at least one label is ferrocene. In this article, the detectable label can serve as a signaling moiety. In some embodiments, the signal primer does not bind / hybridize with the first capture probe. In some embodiments, the first part of the signal amplifier of the single strand is combined with / hybridized with the first capture probe, and the second part of the signal amplifier of the single strand is combined with / hybridized with the second capture probe. In some embodiments, the labeled primer, the double-stranded labeled amplifier and / or the single-stranded labeled amplifier include at least one label selected from the group consisting of ferrocene, methylene blue or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the labeled primer includes a ferrocene label on the 5'-end, on any one of the nucleotides 1 to 3 at the 5'-end, on the 3'-end, on any one of the nucleotides 1 to 3 at the 3'-end, or on both the 5'-end and the 3'-end. In some embodiments, the labeled primer is included in the ferrocene label inside the labeled primer. In some embodiments, the labeled primer, the double-stranded labeled amplifier and / or the single-stranded labeled amplifier include multiple ferrocene labels at at least two positions. In some embodiments, the single-stranded labeled amplicon comprises a first segment capable of hybridizing with a first capture probe, a second segment incapable of binding to the capture probe, and a third segment comprising the at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue, or osmium. In some embodiments, the labeled primer comprises a first segment capable of hybridizing with a first capture probe, a second segment comprising a joint, and a third segment comprising the at least one label, wherein the joint connects the first segment and the third segment. In some embodiments, the method further comprises: separating the labeled primer and the single-stranded labeled amplicon before electrochemical detection. In another embodiment, the method further comprises: separating the labeled primer and the double-stranded labeled amplicon before electrochemical detection. In some embodiments, the labeled primer consists of a first segment capable of hybridizing with a capture probe and a second segment comprising at least the first label.In some embodiments, the amplification reagents include a nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer necessary for the function of the nucleic acid polymerase, and a divalent cation (such as, for example, Mg. 2+ and / or Mn 2+ ). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises the step of using a reverse transcriptase to reverse transcribe the RNA to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity.

[0006] In a third aspect, a method for detecting the presence of a target single-stranded or double-stranded nucleic acid in a sample, the method comprising the following steps: (a) providing (i) a sample suspected of containing the target nucleic acid, (ii) a nucleic acid primer comprising: a nucleic acid sequence complementary to at least a portion of the target nucleic acid, and a first electrochemically detectable label, and (iii) a reagent for extending the nucleic acid chain; (b) forming a reaction mixture comprising the above (i), (ii) and (iii); (c) contacting the nucleic acid primer with the target nucleic acid in the presence of the target nucleic acid under hybridization conditions; (d) extending the nucleic acid primer in the presence of the target nucleic acid, thereby incorporating the electrochemically detectable label into the amplicon to form an electrochemically labeled amplicon; (e) denaturing the electrochemically labeled amplicon; (f) hybridizing the electrochemically labeled amplicon with a capture probe bound to an electrode surface; (g) detecting the presence of the target nucleic acid by detecting energy transfer between the electrochemically labeled amplicon and the electrode surface. In some embodiments, the electrochemically detectable label is selected from the group consisting of ferrocene, methylene blue or osmium. In certain embodiments, the at least one label is ferrocene. In this article, detectable label can serve as signaling part. In certain embodiments, nucleic acid primer comprises a first part that is combined with / hybridized with a capture probe and a second part that is not combined with / hybridized with a capture probe. In certain embodiments, nucleic acid primer further comprises a second electrochemical detectable label, and the first electrochemical detectable label and the second electrochemical detectable label are different. In certain embodiments, nucleic acid primer is a signal primer that is not combined with / hybridized with the first capture probe. In certain embodiments, the first part of the signal amplifier of the single strand is combined with / hybridized with the first capture probe, and the second part of the signal amplifier of the single strand is combined with / hybridized with the second capture probe. In certain embodiments, nucleic acid primer, double-stranded labeled amplifier and / or single-stranded labeled amplifier comprise at least one label selected from the group consisting of ferrocene, methylene blue or osmium. In certain embodiments, the at least one label is ferrocene. In some embodiments, the nucleic acid primer comprises a ferrocene label at the 5'-end, at any one of nucleotides 1 to 3 at the 5'-end, at the 3'-end, at any one of nucleotides 1 to 3 at the 3'-end, or at both the 5'-end and the 3'-end. In some embodiments, the nucleic acid primer comprises a ferrocene label inside the labeled primer. In some embodiments, the nucleic acid primer, the double-stranded labeled amplicon and / or the single-stranded labeled amplicon comprise multiple ferrocene labels at at least two positions. In some embodiments, the single-stranded labeled amplicon comprises a first segment capable of hybridizing with the first capture probe, a second segment that cannot bind to the capture probe, and a third segment comprising the at least one label. In certain embodiments, the label is selected from the group consisting of ferrocene, methylene blue or osmium.In some embodiments, the nucleic acid primer comprises a first segment capable of hybridizing with a first capture probe, a second segment comprising a joint, and a third segment comprising the at least one label, wherein the joint connects the first segment and the third segment. In some embodiments, the method further comprises: separating the nucleic acid primer and the single-stranded labeled amplicon before electrochemical detection. In another embodiment, the method further comprises: separating the nucleic acid primer and the double-stranded labeled amplicon before electrochemical detection. In some embodiments, the nucleic acid primer consists of a first segment capable of hybridizing with a capture probe and a second segment comprising the at least first label. In some embodiments, the reagent for performing nucleic acid chain extension comprises a nucleic acid polymerase, deoxynucleotide triphosphates (dNTPs), a reaction buffer necessary for the function of the nucleic acid polymerase, and a divalent cation (such as, for example, Mg. 2+ and / or Mn 2 + ). In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises the step of using a reverse transcriptase to reverse transcribe the RNA to generate cDNA. In some embodiments, the nucleic acid polymerase is a DNA polymerase. In some embodiments, the nucleic acid polymerase further exhibits reverse transcriptase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1a A typical sandwich assay used in electrochemical detection is shown. Figure 1a In the sandwich structure, three main elements are included: a capture probe (2), a signal probe (3) and a target (4). In a nucleic acid embodiment, a target may be synonymous with a PCR amplicon sequence. The target may have a portion (4b) that specifically binds or hybridizes to a desired portion (3b) of the signal probe, a portion (4a) that binds or hybridizes to a corresponding capture probe portion (2b), and optionally, for example, one or more flanking portions (4c). The capture probe may include a connector (2a) that connects, engages or binds the capture probe (2) to an electrode surface (1). As shown, the signal probe has a detectable labeled portion or label (3a) that is adjacent to the electrode surface. The labeled portion 3a is conjugated to or inside the signal probe binding portion (3b). Not shown is a self-assembled monolayer (SAM), which in some variants is also attached to the electrode surface via one or more connectors in a form similar to the capture probe connector (2a), and which is used to prevent or reduce undesirable electron transfer events ("noise") to the electrode surface.

[0008] Figure 1b A typical signaling complex as disclosed herein is shown. Figure 1bIn the assay, only two main elements are included: a capture probe (2) and a target (4). In nucleic acid embodiments, the target may be synonymous with a PCR amplicon sequence. The target may have a portion (4a) that specifically binds or hybridizes to a desired portion (2b) of the capture probe portion. Not shown is that the target (4) may optionally be included in one or more portions flanking the capture probe. The capture probe may include a connector (2a) that connects, engages or binds the capture probe (2) to the electrode surface (1). As shown, the target has a detectable labeled portion or label (3a) in close proximity to the electrode surface. The labeled portion 3a is conjugated to the target at the signal binding portion (4b). Not shown is a self-assembled monolayer (SAM), which in some embodiments is also attached to the electrode surface via one or more connectors in a form similar to the capture probe connector (2a), and which is used to prevent or reduce undesirable electron transfer events ("noise") to the electrode surface.

[0009] Figure 2 Examples of signal primers disclosed herein are shown. Figure 2 a to Figure 2 c shows different embodiments of signal primers disclosed herein. In some embodiments, the first signal moiety (which is shown as 3 circles) is on the 5'-end of the signal primer, and the second signal moiety is conjugated internally to the signal primer itself ( Figure 2 a). In some embodiments, the signal moiety (which is shown as 3 circles) is on the 5'-end of the signal primer ( Figure 2 c). In some embodiments, the signal moiety (which is shown as 3 circles) is conjugated internally to the signal primer itself ( Figure 2 b) In other words, the redox moiety is attached to the primer and the label can be introduced at the 5'-end or anywhere in the middle of the sequence. Figure 2 In (a to c), "ii" is the detection moiety (eg, ferrocene), and "i" is the annealing region (eg, the region that binds to the DNA template during PCR).

[0010] Figure 2 d to Figure 2 f shows different embodiments of single-stranded signal amplifiers disclosed herein. In some embodiments, the first signal portion (which is shown as 3 circles) is on the 5'-end of the single-stranded signal amplifier, and the second signal portion is conjugated internally to the signal primer itself ( Figure 2 d). In some embodiments, the signal moiety (which is shown as 3 circles) is at the 5'-end of the single-stranded signal amplifier ( Figure 2 f). In some embodiments, the signal moiety (which is shown as 3 circles) is conjugated internally to the single-stranded signal amplifier ( Figure 2e). In other words, the redox moiety is attached to the single-stranded signal amplifier and the label can be introduced at the 5'-end or anywhere in the middle of the sequence. Figure 2 In (d to f), "ii" is the detection moiety (eg, ferrocene), and "i" is the annealing region (eg, the region that binds to the capture probe during detection).

[0011] Figure 2 g to Figure 2 K shows different embodiments of double-stranded signal amplifiers disclosed herein. In some embodiments, the first signal portion (which is shown as 3 circles) is on the 5'-end of the double-stranded signal amplifier, and the second signal portion is conjugated internally to the signal primer itself. ( Figure 2 g). In some embodiments, the signal moiety (which is shown as 3 circles) is at the 5'-end of the single-stranded signal amplifier ( Figure 2 i). In some embodiments, the signal moiety (which is shown as 3 circles) is conjugated to or within the single-stranded signal amplifier ( Figure 2 h).

[0012] Alternatively, the primer, single-stranded signal amplifier or double-stranded signal amplifier may contain additional terminal nucleosides (n+1 or n+2) at the end of the nucleic acid, which are used to covalently attach the electron transfer moiety but do not participate in base pair hybridization during PCR or detection. Figure 2 j shows an additional terminal nucleoside attached to the signal moiety at the 5'-end of the double-stranded signal amplifier, and the second signal moiety is conjugated internally to the signal primer itself. Figure 2 k shows the additional terminal nucleoside attached to the signal moiety at the 5'-end of the double-stranded signal amplifier.

[0013] Alternatively, the primer may contain a linker for covalently attaching the electron transfer moiety. Figure 2 1 shows the linker to which the signal moiety is attached at the 5'-end. Figure 2 m shows a linker attached to the signal moiety at the 3'-end. Figure 2 n shows an embodiment in which the primer contains a signal moiety at both the 5'-end and the 3'-end.

[0014] Figure 3 An embodiment of the system disclosed herein is shown. Figure 3As seen in , the capture probe combined with a detectable label (shown as a circle) can be sampled individually or continuously on the detection site. The capture probe combined with a detectable label can be specific for the target analyte. The capture probe combined with a detectable label can be used as a control, and can contain its own signal, which can be different from the signal combined with the analyte. In other words, the labeled nucleic acid capture probe can have a redox potential that is the same or different from the label on the single-stranded signal amplifier. In some cases, the single-stranded signal amplifier can be combined with the labeled capture probe, and in some cases it cannot be combined. In certain embodiments, the label on the capture probe and the label on the single-stranded signal amplifier are the same, but they do not have to be the same. In certain embodiments, they are energy transfer moieties. In certain embodiments, they are ferrocene-based, ferrocene-derived compounds, methylene blue or osmium.

[0015] Figure 4 An embodiment of the system disclosed herein is shown. Because the amplifier no longer needs to have a portion that binds to the signal probe as in a typical sandwich assay, the amplifier can be shorter. Nevertheless, in some cases, it can be beneficial to amplify longer amplifiers and have multiple capture probes that bind to different portions of the amplifier. The method can utilize one or more capture probes that bind to different portions of the single-stranded signal amplifier on the same detection electrode. In some embodiments, the portion (a) of the single-stranded signal amplifier that binds to the first capture probe (e) does not cross-hybridize with the second capture probe (d). In some embodiments, the portion (b) of the single-stranded signal amplifier that binds to the first capture probe (d) does not cross-hybridize with the second capture probe (e). In some embodiments, the first portion (b) of the single-stranded signal amplifier binds to the first capture probe (d) and does not cross-hybridize with the second capture probe (e) that binds to the second portion (a) of the single-stranded signal amplifier. In some embodiments, the first portion (a) of the single-stranded signal amplifier can bind to the first capture probe (d) and the second capture probe (e), and the second portion (b) of the single-stranded signal amplifier cross-hybridizes with the second capture probe (e) and the first capture probe (d). In some embodiments, the first portion (a) of the single-stranded signal amplifier may bind to both the first capture probe (d) and the second capture probe (e), but the second portion (b) of the single-stranded signal amplifier may bind only to the second capture probe (e).

[0016] Figure 5 An embodiment of the system disclosed herein is shown. Figure 5As seen in , in some embodiments, the single-stranded signal amplifier has three parts. The first part (a) is bound to the capture probe (d), the second part (b) is bound to the signal probe (e), and the third part is bound to the electron transfer part (c). In some embodiments, the single-stranded signal amplifier detectable label has a different potential from the corresponding signal probe. In some embodiments, the single-stranded signal amplifier detectable label has the same potential as the corresponding signal probe. In some embodiments, the portion (a) of the single-stranded signal amplifier that binds the capture probe (d) does not cross-hybridize with the signal probe (e). In some embodiments, the portion (a) of the single-stranded signal amplifier that binds the capture probe cross-hybridizes with the signal probe (b).

[0017] Figure 6 An example of a single-stranded signal amplifier disclosed herein is shown. Figure 6 As seen in , segment c is the region of the single-stranded signal amplifier that can bind to the capture probe. Segment b is the primer region of the single-stranded signal amplifier, and it cannot bind to the capture probe. Segment a is the region of the signaling portion of the single-stranded signal amplifier, which also cannot bind to the capture probe. In some embodiments, segment a is 5 to 100 base pairs away from segment c. In some embodiments, segment a is 5 to 200 base pairs away from segment c. In some embodiments, segment a is 25 to 100 base pairs away from segment c.

[0018] Figure 7 N6 is shown. N6 is a tag that can be used; its synthesis is described in commonly owned U.S. Patent No. 7,393,645, the entire contents of which are incorporated herein by reference.

[0019] Figure 8 QW56 ferrocene label is shown. QW56 and QW80 ferrocene labels can be prepared using conventional DNA synthesis techniques essentially as described in commonly owned application PCT / US08 / 82666 (published as WO / 2009 / 061941A2 and U.S. Pat. No. 7,820,391), the entire contents of which are incorporated herein by reference. In U.S. Pat. No. 9,891,215 (the entire contents of which are incorporated herein by reference), Figure 3 A depicts QW 56 and Figure 3 B depicts QW80.

[0020] Fig. 9 QW80 ferrocene labeling is shown.

[0021] Fig.10 The signal (nA) generated by the SARS-CoV-2 amplicon is shown. Data from two detection pads for three replicates are shown.

[0022] Fig.11 The maximum signal (nA) from all targets in the detection zone is shown.

[0023] Fig.12 The signals (nA) from targets using traditional sandwich assays (control primers) and signal primers to the indicated targets were compared. DETAILED DESCRIPTION

[0024] definition

[0025] As used in the claims and herein, the following terms have the following definitions:

[0026] As used herein, "amplification" refers to any in vitro method for increasing the number of copies of a nucleotide sequence using a polymerase. Nucleic acid amplification causes nucleotides to be incorporated into nucleic acid molecules (e.g., DNA) or primers, thereby forming new nucleic acid molecules complementary to nucleic acid templates. The formed nucleic acid molecules and their templates can be used as templates for synthesizing other nucleic acid molecules. As used herein, an amplification reaction can be composed of multiple rounds of nucleic acid synthesis. Amplification reactions include, for example, polymerase chain reaction (PCR). A PCR reaction can be composed of 5 to 100 nucleic acid molecule denaturation and synthesis "cycles".

[0027] An "amplification primer" or "primer" is a primer used to amplify a target sequence by primer extension.

[0028] An "amplicon" is a nucleic acid molecule that contains a primer or a portion of a primer and a newly synthesized strand that is the complement of the sequence downstream of the primer binding site. Extension products are produced by hybridization of a primer to a template containing a complementary sequence and extension of the primer by a polymerase using the template.

[0029] "Analyze" means to measure, detect, or determine the presence, absence, or composition of something.

[0030] "Analyte" is any substance that can selectively bind to a capture binding ligand. The analyte can be natural, biological or synthetic, for example, any synthetic molecule or other molecule such as that used in drug discovery that exhibits an exceptionally good or specific binding affinity to a "capture binding ligand". Both the analyte and the capture binding ligand can be composed of one or more different domains. The skilled person will appreciate that complementary orientation between the analyte and the capture binding ligand is required. Suitable analytes include organic and inorganic molecules, including biomolecules. In an embodiment, the analyte can be an environmental pollutant (including pesticides, insecticides, toxins, etc.); chemicals (including solvents, organic materials, etc.); therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.); biological molecules (including hormones, cytokines, proteins, lipids, carbohydrates, cell membrane antigens and receptors (neural receptors, hormone receptors, nutrient receptors and cell surface receptors) or their ligands, etc.); whole cells (including prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells (including mammalian tumor cells)); viruses (including retroviruses, herpes viruses, adenoviruses, lentiviruses, etc.); and spores; etc.

[0031] Suitable nucleic acid target analytes include, but are not limited to, any number of viruses (including orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g., smallpox virus, vaccinia virus), enteroviruses (e.g., polio virus, coxsackie virus), hepatitis viruses (including types A, B, and C), herpes viruses (e.g., herpes simplex virus, varicella zoster virus, cytomegalovirus, , Epstein-Barr virus), rotavirus, Norwalk virus, Hantavirus, Arenavirus, Rhabdovirus (e.g., rabies virus), Retrovirus (including HIV, HTLV-I and II), Papovavirus (e.g., papillomavirus), Polyomavirus and Picornavirus, etc.), and Bacteria (including a wide variety of target pathogenic and non-pathogenic prokaryotes, including Bacillus; Vibrio, such as Vibrio cholerae; Escherichia, such as Enterotoxigenic Escherichia coli E. coli), Shigella, for example, Shigella dysenteriae (S. dysenteriae); Salmonella, for example, Salmonella typhi (S. typhi); Mycobacterium, for example, Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium leprae (M. leprae); Clostridium, for example, Clostridium botulinum (C. botulinum), Clostridium tetani (C. tetani), Clostridium difficile (C. dificile), Clostridium perfringens (C. perfringens); Corynebacterium, for example, Corynebacterium diphtheriae diphtheriae; Streptococci, such as S.pyogenes and S.pneumoniae; Staphylococci, such as S.aureus; Haemophilus, such as H.influenzae; Neisseria, such as N.meningitidis and N.gonorrhoeae; Yersinia, such as G.lamblia and Yersinia pestis. pestis), Pseudomonas, e.g., Pseudomonas aeruginosa, Pseudomonas putida; Chlamydia, e.g., Chlamydia trachomatis; Bordetella, e.g., Bordetella pertussis; Treponema, e.g., Treponema pallidum; etc.) (collectively referred to as "bacterial and viral targets") nucleic acids.

[0032] Suitable nucleic acid target analytes include, but are not limited to, any number of Gram-positive organisms (including Bacillus cereus group, Bacillus subtilis group, Corynebacterium spp., Propionibacterium acnes, Propionibacterium acnes, Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, Staphylococcus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus, Streptococcus agalactiae (GBS), Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus pyogenes) (GAS), resistance genes, mecA, mecC, vanA or vanB) (collectively referred to as "Gram-positive targets").

[0033] Suitable nucleic acid target analytes include, but are not limited to, any number of Gram-negative organisms (including Acinetobacter baumannii, Bacteroides fragilis, Citrobacter rodentium, Cronobacter sakazakii, Enterobacter (non-cloacae complex), Enterobacter cloacae complex, Escherichia coli, Fusobacterium nucleatum, Fusobacterium necrophorum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomonas maltophilia, resistance genes, CTX-M, IMP, KPC, NDM, OXA (OXA-23 and OXA-48), or VIM (collectively referred to as "Gram-negative targets")).

[0034] Suitable nucleic acid target analytes include, but are not limited to, nucleic acids from any number of fungal organisms, including Candida albicans, Candida auris, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus gattii, Cryptococcus nehoformans, Fusarium, or Rhodotorula (collectively, "fungal targets").

[0035] In some embodiments, the target is a human-specific infectious disease pathogen or target, wherein the marker or target is a nucleic acid marker.

[0036] "Array" means a plurality of distinct sites with different capture binding ligands. In some embodiments, the array is "addressable" to the extent that the individual sites have predetermined or determinable positions relative to each other, optionally with the aid of electronic connectors and / or software.

[0037] "Capture binding ligand" is synonymous with "capture probe" or "capture binding probe" and is a compound that exhibits a relatively strong or specific affinity for another compound such that it is able to extract that other compound away from a group of other compounds in a mixture of compounds. The capture binding ligand can be a protein, carbohydrate, nucleic acid, small molecule, or any combination of these.

[0038] "Double-stranded signal amplifier" means a double-stranded amplifier formed during PCR by using primers containing an electrochemically detectable label. The double-stranded signal amplifier contains a nucleic acid and a signal primer.

[0039] "Electrode" means a composition that can sense current or charge and convert it into a signal when connected to an electronic device. Electrodes are known in the art and include, but are not limited to, certain metals and their oxides, including gold; platinum; palladium; silicon; aluminum; metal oxide electrodes (including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (WO3), and ruthenium oxide); and carbon (including glassy carbon electrodes, graphite, and carbon paste).

[0040] "Electrochemical detection" means using at least two electrodes to apply a potential and measure the current generated by a chemical reaction. For clarity, "electrochemical detection" does not include (i) detection of the conductivity, impedance, or capacitance of a droplet, a portion of a droplet, or the contents of a droplet; (ii) detection by electrochemiluminescence; and (iii) detection by optical means.

[0041] The terms "electron donor moiety," "electron acceptor moiety," and "electron transfer moiety," or grammatical equivalents herein, refer to molecules that are capable of electron transfer under certain conditions. It should be understood that electron donor and acceptor capabilities are relative; that is, a molecule that can lose electrons under certain experimental conditions will be able to accept electrons under different experimental conditions. It should be understood that the number of possible electron donor moieties and electron acceptor moieties is very large, and a technician in the field of electron transfer compounds will be able to utilize a variety of compounds and the selection of those compounds is within the skill of the technician. One advantage of redox-mediated electron detection is that there are a variety of different electron transfer moiety labels, each with its own distinguished potential that can be selectively measured or filtered. Some electron transfer moieties include, but are not limited to, transition metal complexes, organic electron transfer moieties, electrodes, metallocene complexes such as ferrocene and ferrocene derivatives, or methylene blue or osmium.

[0042] As used herein, the terms "hybridization" and "hybridizing" refer to the pairing of two complementary single-stranded nucleic acid molecules (RNA and / or DNA) to produce a double-stranded molecule. As used herein, two nucleic acid molecules can hybridize despite incomplete base pairing complementarity. Therefore, mismatched bases will not prevent the hybridization of two nucleic acid molecules, provided that appropriate conditions well known in the art are used.

[0043] The term "fixed" or derivatives thereof includes attached, associated, or bound, whether covalently or non-covalently.

[0044] "Infectious disease" means a disease marked by an infectious disease marker, pathogen, or target, whether viral, bacterial, or fungal. Exemplary infectious disease targets include, for example, natural, synthetic, or amplified biomolecules such as: viral targets, Gram-negative targets, Gram-positive targets, and fungal targets.

[0045] "Label" means something that can signal or be stimulated to signal an event or the presence of a molecule or molecular complex. Labels can include, for example, dyes, radioactive atoms or molecules, redox-active compounds, enzymes, enzyme substrates, nucleic acids, their derivatives, etc. Similar to dyes and chemiluminescent compounds that exist in different colors, redox-active labels have a variety of different potentials that can be used.

[0046] "Monolayer" or "self-assembled monolayer" or "SAM" herein means a relatively ordered assembly of molecules spontaneously chemisorbed on a surface, wherein the molecules are oriented generally parallel to each other and generally perpendicular to the surface. Each of the molecules comprises a functional group that adheres to the surface, as well as a portion that interacts with adjacent molecules in the monolayer to form a relatively ordered array. "Mixed" monolayers include heterogeneous monolayers, i.e., where at least two different molecules make up the monolayer.

[0047] "Nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. Nucleic acids generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs that may have alternating backbones (including, for example, phosphoramide) are included. Nucleic acids can be DNA (both genomic and cDNA), RNA, or hybrids, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides and any combination of bases (including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc.). As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, as well as modified nucleosides (such as amino-modified nucleosides). In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, for example, individual units of peptide nucleic acids (each of which contains a base) are referred to herein as nucleosides.

[0048] As used herein, "nucleotide" refers to a base-sugar-phosphate combination. Nucleotide is a monomer unit of a nucleic acid sequence (DNA and RNA). The term nucleotide includes monophosphate, diphosphate and triphosphate forms of deoxyribonucleosides and ribonucleosides and their derivatives. The term nucleotide particularly includes deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or their derivatives. Such derivatives include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP. As used herein, the term nucleotide also refers to dideoxyribonucleoside triphosphates (ddNTP) and their derivatives. The illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddITP. As used herein, nucleotides are unlabeled.

[0049] As used herein, "polymerase" refers to any enzyme having nucleotide polymerization activity. Polymerases (including DNA polymerases and RNA polymerases) include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or ) DNA polymerase, Pyrococcus furious (Pflu) DNA polymerase, DEEPVENT DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME) DNA polymerase, Methanobacterium thermoautotrophicum) (Mth) DNA polymerase, mycobacterium DNA polymerase (Mtb.Mlep) and their mutants, variants and derivatives. RNA polymerases such as T3, T5 and SP6 and their mutants, variants and derivatives can also be used.

[0050] As used herein, "primer" refers to a synthetic or biologically produced single-stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule. Nucleic acid amplification is usually based on nucleic acid synthesis performed by nucleic acid polymerase or reverse transcriptase. Many such polymerases or reverse transcriptases require the presence of primers that can be extended to initiate the synthesis of such nucleic acids.

[0051] As used herein, "probes" refer to synthetic or biologically produced nucleic acids (DNA or RNA) that are designed or selected to contain specific nucleotide sequences that allow them to hybridize specifically (ie, preferentially) to a target nucleic acid sequence under defined stringency.

[0052] A "redox active" compound or moiety means a compound or moiety that is capable of transferring, shuttling, or accepting electrons from another redox active compound. Some redox active compounds include electrodes and metallocene complexes (including ferrocene and its derivatives), methylene blue, or osmium.

[0053] A "sample solution" may contain any number of substances, including but not limited to body fluids (including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen from virtually any organism (such as mammalian samples, including human samples)); environmental samples (including but not limited to air samples, agricultural samples, water samples, and soil samples); biological warfare agent samples; research samples (i.e., in the case of nucleic acids, the sample can be the product of an amplification reaction, including both target and signal amplification (such as a PCR amplification reaction)); purified samples, such as purified genomic DNA, RNA, proteins, etc.; original samples (bacteria, viruses, genomic DNA), etc.; as will be understood by those skilled in the art, the sample can be subjected to virtually any experimental manipulation.

[0054] "Signal primer" means a primer comprising an electrochemically detectable label.

[0055] "Signaling probe" means a probe molecule with a label that binds to the analyte and signals the presence of the analyte. For electrochemical detection, the label is usually ferrocene or a ferrocene derivative, which binds to one domain of the analyte, while another domain of the analyte binds to a capture binding ligand on a site on the surface of a solid support (a configuration known as a "sandwich assay").

[0056] "Single-stranded signal amplifier" means an amplifier formed during PCR by using a primer comprising an electrochemically detectable label. The single-stranded signal amplifier comprises a nucleic acid and a signal primer.

[0057] A "solid support" or "support" can be any material or matrix suitable for attaching oligonucleotides / capture probes. Such oligonucleotides and / or capture probes can be added to or bound to a support (covalently or non-covalently) by any technique or any combination of techniques well known in the art. The support can be any substance other than an aqueous phase at room temperature, and includes, for example, beads, gels, columns, column matrices, multi-titer plates, paper, membranes, printed circuit boards, or other array surfaces or supports.

[0058] The term "target nucleic acid" or "target" or grammatical equivalents herein refers to a nucleic acid sequence to be amplified or detected. These include the original nucleic acid sequence to be amplified, its complementary second strand, and any strand of a copy of the original sequence produced by replication or amplification. The target sequence may also be referred to as a template for extending a hybridization primer.

[0059] The target sequence can be a part of a gene, a regulatory sequence, a genomic DNA, a cDNA, an RNA (including mRNA and rRNA), or other. It can be of any length, wherein it should be understood that the longer the sequence, the greater the specificity. As will be appreciated by those skilled in the art, the complementary target sequence can take a variety of forms. For example, it can be contained within a larger nucleic acid sequence (i.e., all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, etc.). As more fully outlined below, the probe is hybridized with the target sequence to determine the presence or absence of the target sequence in the sample. The target sequence can also be composed of different target domains; for example, the first target domain of the sample target sequence can hybridize with a capture probe or a portion of the capture probe, and the second target domain can hybridize with a portion of a different capture probe. The target domains can be adjacent or separated. The terms "first" and "second" do not mean to confer an orientation relative to the 5'-3' orientation of the target sequence. For example, assuming a 5'-3' orientation of the complementary target sequence, the first target domain can be located at the 5' of the second domain or the 3' of the second domain. A target refers to a nucleic acid molecule to which a specific primer or probe can preferentially hybridize.

[0060] As used herein, "target sequence" refers to a nucleic acid sequence within a target molecule to which a specific primer or probe can preferentially hybridize.

[0061] As used herein, the term "template" refers to a double-stranded or single-stranded molecule to be amplified, synthesized or sequenced. In the case of a double-stranded DNA molecule, denaturation is performed to form a first chain and a second chain to amplify, sequence or synthesize these molecules. A primer complementary to a part of the template is hybridized under appropriate conditions, and then a polymerase (DNA polymerase or reverse transcriptase) can synthesize a nucleic acid molecule complementary to the template or a part thereof. The length of the newly synthesized molecule can be equal to or shorter than the original template. Mismatches during the synthesis or extension of the newly synthesized molecule are incorporated to cause one or more mismatched base pairs. Therefore, the synthesized molecule does not need to be fully complementary to the template. The template can be an RNA molecule, a DNA molecule or an RNA / DNA hybrid molecule. The newly synthesized molecule can be used as a template for subsequent nucleic acid synthesis or amplification.

[0062] As used herein, recombinant DNA technology and other terms used in the fields of molecular and cellular biology will be commonly understood by those of ordinary skill in the applicable arts.

[0063] General description of the apparatus and method

[0064] Disclosed are devices and methods for nucleic acid amplification and detection. The device can be configured for electrochemical detection methods. Disclosed are methods and reagents for amplifying nucleic acid sequences and their use in nucleic acid detection.

[0065] In a first embodiment, an oligonucleotide primer (i.e., a signal primer) used in a nucleic acid amplification assay is disclosed, comprising a primer region and an electron transfer moiety (ETM) (e.g., a ferrocene label). In some embodiments, the primer does not comprise a secondary structure in solution at any temperature. In some embodiments, the primer is capable of binding to a target nucleic acid at any temperature. In some embodiments, the oligonucleotide primer is self-annealing.

[0066] Therefore, in a first aspect, a method for amplifying one or more nucleic acid molecules is provided, the method comprising: (a) mixing one or more template or target nucleic acid molecules or a sample suspected of containing a target nucleic acid with one or more signal primers described herein and reagents necessary for amplification; and (b) incubating the mixture under conditions sufficient to amplify one or more nucleic acid molecules complementary to all or a portion of the template or target molecule. The amplified nucleic acid molecules contain one or more signal primers or portions thereof disclosed herein. In one aspect, an electron transfer moiety (ETM) is incorporated at or near one or both ends of a synthetic or amplified nucleic acid molecule produced by amplification. In one aspect, an electron transfer moiety (ETM) is incorporated at or near the 5'-end.

[0067] On the other hand, the method includes a method for performing a nucleic acid amplification assay, comprising: (a) combining a reagent for nucleic acid amplification, a nucleic acid polymerase, a target nucleic acid or a sample suspected of containing a target nucleic acid and a modified primer, wherein the modified primer comprises a detectable label, wherein the detectable label is ferrocene; (b) circulating the mixture of (a) to provide multiple copies of the amplicon incorporated into the modified primer in the presence of the target nucleic acid in the sample; (c) denaturing the amplicon into a single-stranded signal amplicon; (d) exposing the single-stranded signal amplicon to a capture oligonucleotide complementary to the single-stranded signal amplicon; (e) hybridizing the hybridization region of the single-stranded signal amplicon with the capture oligonucleotide, and (f) detecting the label associated with the hybridization. In some embodiments, detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises a step of reverse transcribing the RNA to generate cDNA using a reverse transcriptase. In some embodiments, the nucleic acid polymerase is a DNA polymerase.

[0068] In yet another embodiment, the method includes a method for performing a nucleic acid amplification assay, comprising: (a) combining a reagent, a nucleic acid polymerase, a target nucleic acid or a sample suspected of containing a target nucleic acid, a modified primer and a second primer, wherein the modified primer comprises a detectable label, wherein the detectable label is ferrocene, and the second primer comprises a base sequence complementary to a second region of the target nucleic acid and does not have a detectable label; (b) cycling the mixture of (a) to provide multiple copies of a double-stranded amplicon incorporating the modified primer and the second primer in the presence of the target nucleic acid in the sample; (c) cycling the mixture of (a) to provide multiple copies of a double-stranded amplicon incorporating the modified primer and the second primer; and (d) cycling the mixture of (a) to provide multiple copies of a double-stranded amplicon incorporating the modified primer and the second primer in the presence of the target nucleic acid in the sample. (d) exposing the signal amplifier of the single strand of the modified primer to a capture oligonucleotide complementary to the signal amplifier of the single strand, and exposing the second single strand of the amplifier to a capture oligonucleotide complementary to the second single strand of the amplifier; (e) hybridizing the signal amplifier of the single strand of the modified primer with the capture oligonucleotide, and hybridizing the second single strand of the amplifier with the capture oligonucleotide; and (f) detecting the label associated with the first primer incorporated into the amplifier. In some embodiments, detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further comprises the step of reverse transcribing RNA to generate cDNA using a reverse transcriptase. In some embodiments, the nucleic acid polymerase is a DNA polymerase.

[0069] In yet another aspect, the method includes a method for performing a nucleic acid amplification assay, comprising: (a) combining a reagent, a polymerase, a target nucleic acid or a sample suspected of containing a target nucleic acid, a first modified primer, and a second modified primer, wherein the first modified primer comprises a first detectable label and the second modified primer comprises a second detectable label, wherein the first detectable label and the second detectable label are ferrocene, and the second modified primer comprises a base sequence complementary to a second region of the target nucleic acid; (b) cycling the mixture of (a) to provide multiple copies of a double-stranded amplicon incorporated with the first modified primer and the second modified primer in the presence of the target nucleic acid in the sample; (c) converting the amplicon incorporated with the first modified primer into a double-stranded amplicon; and (d) cycling the amplicon. (d) exposing the single-stranded signal amplifier incorporated into the first modified primer to a capture oligonucleotide complementary to the first single-stranded signal amplifier, and exposing the second single-stranded signal amplifier to a capture oligonucleotide complementary to the second single-stranded signal amplifier; (e) hybridizing the first single-stranded signal amplifier incorporated into the first modified primer with the first capture oligonucleotide, and hybridizing the second single-stranded signal amplifier with the second capture oligonucleotide; and (f) detecting the label associated with the first primer incorporated into the amplifier, and detecting the label associated with the second primer incorporated into the amplifier. In some embodiments, detection is electrochemical detection. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nucleic acid is RNA, and the method further includes a step of reverse transcribing RNA to generate cDNA using a reverse transcriptase. In some embodiments, the polymerase is a DNA polymerase.

[0070] System Overview

[0071] Current electrochemical detection assays are based on the principle of nucleic acid hybridization using a sandwich assay format, in which a single-stranded amplicon is bound to a sequence-specific signal probe, and the signal probe / single-stranded amplicon complex ("signal probe complex") is bound to a capture probe bound to an electrode, such as Figure 1a , which is a schematic diagram showing signal generation using the current capture probe / signal probe sandwich technique.

[0072] In contrast, provided herein are methods and systems that do not require signaling probes and signaling probe complexes. The disclosed methods and systems can be used for genotyping, subtyping, isotyping, or expression assays.

[0073] First, the target nucleic acid is extracted from the sample, and the target nucleic acid is amplified using a polymerase chain reaction (PCR) using a signal primer to form a double-stranded signal DNA. In some embodiments, the target nucleic acid is DNA, and a primer pair comprising at least one signal primer is directly amplified. In some embodiments, the target nucleic acid can be RNA, and before amplification using a signal primer using a polymerase chain reaction (PCR), it is subjected to reverse transcription amplification to form a double-stranded signal DNA. In this article, the primer for reverse transcription can be another reverse transcription primer or can be a signal primer. In some cases, reverse transcription and amplification reactions can be performed in a one-step reaction (RT-amplification). The resulting double-stranded signal DNA is then digested by exonuclease to form a single-stranded signal DNA. The combination of the single-stranded signal DNA and the capture probe brings the electron transfer part ETM (e.g., ferrocene labeling) to the vicinity of the surface of the gold electrode. When a voltage is applied to the system, an electrical signal ( Figure 1b ). Sequential analysis of each electrode allows detection of multiple analyte targets. The detection instrument measures and interprets the electrical output (in nanoamperes nA) to determine the result for each target nucleic acid (e.g., qualitatively determined as "detected" or "not detected").

[0074] Herein, at least one "signal primer" as provided herein is used to amplify the target nucleic acid. In some embodiments, the signal primer is designed to have a ferrocene label (such as, for example, N6, QW56 or QW80) at or near the 5'-end of the non-phosphorylated oligonucleotide primer (e.g., attached to any one of nucleotides 1 to 3 at the 5'-end, or attached to any one of nucleotides 1 to 2 at the 5'-end). In some embodiments, the amplicon generated by using the signal primer has a ferrocene label on or near its 5'-end (e.g., within nucleotides 1 to 3 or nucleotides 1 to 2 at its 5'-end). As Figure 1b As shown in , when voltage is applied, the single-stranded signal amplifier will bind to the capture probe and bring the ferrocene label to the surface of the gold electrode to generate a signal. Therefore, the signal probe is no longer required for detection.

[0075] Prior to the present disclosure, it was not known whether nucleic acid amplification could occur using primers coupled to a ferrocene label. For example, it was not known whether a polymerase would recognize a primer having an ETM (such as, for example, a ferrocene label) attached thereto. It was also not known whether an ETM (such as, for example, a ferrocene label) bound to a primer could withstand thermal cycling. For example, it was not known whether an ETM (such as, for example, a ferrocene label) itself could withstand thermal cycling. It was also not known whether a Taq polymerase or reverse transcriptase would be inhibited by an ETM (such as, for example, a ferrocene label). In addition, prior to the present disclosure, it was not known whether signal detection could occur using primers coupled to an ETM (such as, for example, a ferrocene label) because it was not known whether a detectable label could be held close enough to an electrode to detect a signal.

[0076] For example, in a conventional prior art sandwich assay, an ETM (such as, for example, a ferrocene label) is attached to a signal probe and the signal probe is bound to the amplicon. In this article, the gap between the signal probe binding site and the capture probe binding site is typically zero bases. Alternatively, the sequence gap between the signal probe and the capture probe can be zero to two bases. In addition, it is known that for conventional sandwich assay systems, if an ETM (such as a ferrocene label) is 5 or more bases away from the capture probe, the signal is dramatically reduced.

[0077] If the ferrocene label is attached to the 5'-end of the amplicon or near the 5'-end of the amplicon (e.g., within nucleotides 1 to 3 or nucleotides 1 to 2 of its 5'-end), as in some embodiments of the present disclosure, it will be farther away from the capture probe (up to 100 base pairs away in some cases), and therefore, the skilled person will not know whether such "distant" ferrocene label will be able to generate a detectable signal. Therefore, provided herein is a capture probe signal primer complex, wherein the ETM (such as, for example, a ferrocene label) is greater than 5 base pairs away from the position where the capture probe hybridizes with it. In some embodiments, a capture probe signal primer complex is provided, wherein the ETM (such as, for example, a ferrocene label) is greater than 10 base pairs away from the position where the capture probe hybridizes with it. In a further embodiment, provided herein is a capture probe signal primer complex, wherein the ETM (such as, for example, a ferrocene label) is greater than 50 base pairs away from the position where the capture probe hybridizes with it. In a further embodiment, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is greater than 100 base pairs away from the position of capture probe hybridization. In a further embodiment, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is greater than 200 base pairs away from the position of capture probe hybridization. In some embodiments, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is between 5 to 100 base pairs away from the position of capture probe hybridization. In a further embodiment, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is between 5 to 500 base pairs away from the position of capture probe hybridization. In certain embodiments, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is between 5 to 50 base pairs away from the position of capture probe hybridization.

[0078] Electrochemical detection system

[0079] Further provided herein is an electrochemical detection system that specifically detects target nucleic acid fragments. The system utilizes a capture probe (such as a nucleic acid or peptide nucleic acid probe) that is complementary and specifically hybridized to a target nucleic acid fragment bound to a detectable label to generate a measurable current when an amperometric potential is applied. In certain embodiments, the electrochemical signal is proportional to the amount of the target nucleic acid in the sample.

[0080] Also provided are compositions and methods for detecting target analytes in samples. Signal primers, single-stranded signal amplifiers, and hybridization complexes combined with single-stranded signal amplifiers provide new and useful ways to detect target analytes. In this article, signal primers that can be used to amplify target nucleic acids include nucleic acids that can specifically hybridize with target nucleic acids, and electrochemically detectable signaling moieties. Therefore, after amplification using signal primers, single-stranded signal amplifiers include nucleic acids and electrochemically detectable signaling moieties. Determination complexes include capture binding ligands attached to electrodes (e.g., gold electrodes) and single-stranded signal amplifiers combined with capture binding ligands, wherein single-stranded signal amplifiers include nucleic acids and electrochemically detectable signaling moieties.

[0081] Signal primers simplify assay development

[0082] Electrochemical detection systems utilizing signal primers are described herein. Conventional prior art electrochemical detection systems utilize sandwich assays for detection. Therefore, the amplicon must be long enough to bind to both the capture probe and the signal probe. In contrast, the use of a signal primer as disclosed herein reduces the length of the amplicon because the need to bind the signal probe is eliminated. This makes the design of primers easier because (1) a shorter region is required to distinguish the target nucleic acid; and (2) unlike conventional sandwich assays in which four conserved regions (forward primer, reverse primer, signal probe, and capture probe) are required, only three conserved regions (forward primer, reverse primer, and capture probe) are required in the case of a signal primer as disclosed herein. In addition, the use of an electrochemical detection moiety that is combined with a nucleic acid amplicon eliminates the need to reduce background signals or enhance signal-to-noise ratios. Specifically, there is no need to include a donor / quencher dye pair, i.e., a fluorescent donor dye or quencher for a donor fluorophore.

[0083] Signal primers improve sensitivity

[0084] Improved sensitivity has been observed with the use of signal primers. Without being bound by any particular theory, in prior art sandwich assays, if the amplicon does not bind to the signal probe with 100% efficiency, the sensitivity of the assay is negatively affected. By eliminating the requirement for the amplicon to bind to the signal probe, the sensitivity of the assay can be improved. It is also believed that the inclusion of an ETM (such as, for example, a ferrocene label) makes it less likely that the signal primer will be misdirected to the target nucleic acid molecule without hindering amplification. Improvements in sensitivity are particularly important for the amplification of template nucleic acids that are difficult to amplify and produce little or no desired amplification product in a PCR reaction.

[0085] Signal primers reduce time to results

[0086] Recent studies have shown that patients with severe sepsis or septic shock show an increased likelihood of death of 7.6% per hour without antibiotic therapy. Liang et al., Empiric Antimicrobial Therapy in Severe Sepsis and Septic Shock: Optimizing Pathogen Clearance, Curr Infect Dis Rep. 2015 Jul; 17(7): 493. If the detection system is performed faster, the survival rate may increase.

[0087] As described above, when amplification is performed using a signal primer, the amplicon does not require a signal probe binding region, and therefore, the amplicon can be shorter. A shorter amplicon means that the time required for amplification is reduced. Reducing the amplification time reduces the time to obtain results, which in turn can save the patient's life.

[0088] Additionally, in prior art sandwich assays, the amplicon must first bind to the signal probe during a hybridization reaction, and then the signal probe-amplicon complex binds to the capture probe. The use of a signal primer eliminates the need for a separate signal probe / amplicon hybridization step, further reducing the time to results.

[0089] Signal primers simplify manufacturing process

[0090] Eliminating the need for a signaling probe simplifies the detection hybridization complex from three components to two, which in turn simplifies the manufacturing process for diagnostic tests / kits.

[0091] The difference over existing technology

[0092] Certain types of signal primers are known in the art (see, for example, U.S. Patent Nos. 8,323,929, 9,068,948 and U.S. Publication No. 20190024167). However, these prior art signal primers all utilize fluorescent labels. In contrast, the signal transduction primers provided herein use labels that can be detected by electrochemical detection. This difference is significant because before the present disclosure, it was not known whether nucleic acid (e.g., DNA) could be amplified in the presence of ETM (such as, for example, ferrocene labeling). For example, it is not known whether Taq polymerase or reverse transcriptase will be inhibited by ETM (such as, for example, ferrocene labeling). More importantly, it is not known whether single-stranded signal amplifiers can be detected when combined with capture probes. This is due to the distance of ETM (such as, for example, ferrocene labeling) from the capture probe. In a traditional sandwich assay system, when the signal probe moves away from the capture probe, the signal decreases sharply.

[0093] This embodiment does not use a reporter probe as disclosed in U.S. Patent No. 8,323,929, the 3' end of which hybridizes with the complement of the 5' adapter sequence of the signal primer to produce a 5' overhang. In addition, this embodiment does not use a polymerase to fill in the overhang and synthesize the complement of the 5' overhang of the reporter probe. This embodiment does not directly or indirectly detect the synthesis of the complement of the reporter probe as an indication of the presence of the target nucleic acid.

[0094] Further, the present embodiment does not detect the presence or amount of target nucleic acid by detecting energy transfer between a first energy transfer element in a nucleic acid primer and a second energy transfer element in an incorporated labeled nucleotide as disclosed in U.S. Pat. No. 9,068,948. The present embodiment does not detect energy transfer between labeled primers, energy transfer between labeled primers and nucleotides, energy transfer between labeled nucleotides, energy transfer between fluorescent intercalators and labeled primers or nucleotides, or energy transfer between labeled primers and nucleotides. The present embodiment does not rely on proximity between primers or nucleic acid constructs on each strand as in U.S. Pat. No. 9,068,948.

[0095] Further, the signal primer of this embodiment is not fixed as in U.S. Publication No. 20190024167. In this embodiment, it is not necessary to reduce or quench the energy emitted from the label or other detectable properties from the label as in U.S. Publication No. 20190024167.

[0096] This system does not allow real-time detection. Unlike fluorescence-based systems (wherein the label can be monitored while the reaction is taking place, i.e., "in real time"), the disclosed signal primer must be combined with the capture probe to detect when the amplification ends. This is the so-called "end point" detection system. Therefore, this system cannot be used to provide semi-quantitative or quantitative information about the initial amount of the target present. However, it is expected that the signal generation is specific and proportional to the presence of the appropriate target molecule in the sample. Further, the signal primer provided herein needs to be hybridized with the capture probe to detect and cannot be detected directly from the PCR product.

[0097] General background on the value of nucleic acid testing

[0098] The applications for nucleic acid testing are vast. Most of the current commercial tests address infectious diseases, including chlamydia, gonorrhea, hepatitis, and human immunodeficiency virus (HIV) viral load; inherited diseases, including cystic fibrosis; coagulation and hematological factors, including hemochromatosis; and cancer, including genes for breast cancer. Other target areas include cardiovascular disease and drug resistance screening (known as pharmacogenomics). Most of the testing is currently performed in centralized laboratories that process hundreds or even thousands of samples per day.

[0099] Sequence-specific hybridization of labeled oligonucleotide probes has long been used as a means for detecting and identifying selected nucleotide sequences. Conventional detection methods for the final step in nucleic acid analysis are well known in the art and include sandwich-type capture methods based on radioactivity, colorimetry, fluorescence, fluorescence resonance energy transfer (FRET), and electrochemistry.

[0100] Labeling the probe with ferrocene provides a relatively sensitive way to facilitate detection of probe hybridization. Some patents address electrochemical detection of nucleic acids, such as U.S. Pat. No. 10,001,476 (the entire contents of which are incorporated by reference) discloses detection using capture probe controls and various capture and signal probe configurations and combinations of configurations that can facilitate accurate and efficient multiplex analyte detection. U.S. Pat. No. 10,001,476 discloses single-stranded DNA on an electrode (capture probe) bound to a target. Such systems require that the ferrocene label be maintained adjacent to the electrode to work. In order to achieve the correct orientation of the ferrocene label relative to the electrode, sandwich assays have been used in the prior art. As described above, sandwich assays require an amplicon that can bind to both the capture probe and the signal probe. It is very complicated to develop primers to amplify unique conserved sequences that can bind both the capture probe and the signal probe. Therefore, it is necessary to develop a simpler system.

[0101] Electrochemical detection

[0102] The method and system provided herein include forming a hybrid complex on a detection electrode under certain conditions, wherein the signal amplifier with a single strand specific to the nucleic acid capture probe is kept in close proximity to the detection electrode and can be detected. Therefore, the method provided herein includes testing the presence of a hybrid complex on an electrode under certain conditions, wherein the signal amplifier with a single strand combined with the capture probe is in close proximity to the electrode and transfers electrons thereto. The signal amplifier and signal primer with a single strand not combined with the capture probe are driven away from the electrode and are not detected when the voltage / current is turned on.

[0103] In some embodiments, the detection method is implemented using electrochemical detection in a disposable cartridge. In some embodiments, the electrochemical detection comprises an electrode and a capture binding ligand, the electrode comprising a monolayer, the monolayer comprising a conductive oligomer.

[0104] In some embodiments, the method can utilize one or more capture probes that are combined with different parts of the signal amplifier of the single strand on the same detection electrode. When multiple capture probes are used, each of them has specificity for different parts of the common target nucleic acid sequence. The target nucleic acid sequence can be amplified nucleic acid (e.g., by PCR). In some embodiments, the electrode has a self-assembled monolayer ("SAM"). In some embodiments, the electrode has a mixed SAM of two or more substances, each of which is characterized by different chain lengths, conjugated bond numbers (if any) and / or substituents (if any).

[0105] In some embodiments, the ETM is a detectable label, such as a ferrocene label. In some embodiments, the ferrocene label is selected from the group consisting of N6, QW56, and QW80. In certain embodiments, the detectable label is N6 (see, e.g., Figure 7 In certain embodiments, the detectable marker is QW56 (see, e.g. Figure 8 In certain embodiments, the detectable marker is QW80 (see, e.g. Fig. 9 ). In this context, the detection method is electrochemical.

[0106] In certain embodiments, the capture oligonucleotide is fixed on a gold surface. In other embodiments, the capture oligonucleotide is fixed on an electrode. In some embodiments, a single-stranded signal amplifier comprising a modified signal primer is hybridized with the capture oligonucleotide. In some embodiments, the label used is an electron transfer moiety (ETM), and the addressable solid support detection site is a detection electrode spotted with a capture probe. In some embodiments (e.g., an electrochemical detection embodiment of a nucleic acid analyte using a detection electrode), the site is also characterized by an insulating self-assembled monolayer or a mixed monolayer.

[0107] Electrochemical detection is known to the skilled person. Generally, at least a first input signal is applied to the assay complex and an output signal is received. The output signal is then processed to detect the presence of the target analyte. Some embodiments utilize multiple assay complexes, each of which is attached to a different cell or pad of an array.

[0108] Hybridization

[0109] In the above PCR reaction, the amplicon is generated by two amplification primers, which generate two different single-stranded regions. After amplification, each amplified single-stranded signal amplicon can bind to the capture probe and generate a signal.

[0110] Electronic delivery

[0111] In some embodiments, the detection of ETM is based on electron transfer through the stacked π-orbitals of double-stranded nucleic acids. This basic mechanism is described in U.S. Patent Nos. 5,591,578, 5,770,369, 5,705,348 and PCT US97 / 20014 (all of which are incorporated herein by reference). In short, previous work has shown that electron transfer can be carried out quickly through the stacked π-orbitals of double-stranded nucleic acids and significantly slower through single-stranded nucleic acids. Therefore, this can be used as the basis for determination. Therefore, by adding ETM to nucleic acids attached to detection electrodes via conductive oligomers, electron transfer between ETM and electrodes carried out by nucleic acids and conductive oligomers can be detected.

[0112] Detection electrode

[0113] In an embodiment, the detection electrodes are formed on a substrate that is typically formed of gold electrodes. However, as will be appreciated by those skilled in the art, other electrodes may also be used. The substrate may comprise a variety of materials, as will be appreciated by those skilled in the art. In one embodiment, the substrate comprises a printed circuit board (PCB). Thus, in general, suitable substrates include, but are not limited to, fiberglass, Teflon, ceramic, glass, silicon, mica, plastics (including acrylic resins, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethane, Teflon, TM and their derivatives, etc.), GETEK (a blend of polypropylene oxide and glass fiber), etc.

[0114] In general, materials include printed circuit board materials. Circuit board materials are those materials that include an insulating substrate that is coated with a conductive layer and processed using lithography techniques, particularly photolithography techniques, to form patterns of electrodes and interconnects (sometimes referred to as interconnects or leads in the art). The insulating substrate is typically, but not always, a polymer. As is known in the art, one or more layers can be used to prepare a "two-dimensional" (e.g., all electrodes and interconnects in a plane) or a "three-dimensional" (wherein the electrodes are on one surface and the interconnects can pass through the board to the other side) board. Three-dimensional systems often rely on the use of drilling or etching, followed by electroplating with a metal (such as copper) to make a "throughboard" interconnect. Circuit board materials are typically provided with a foil (such as a copper foil) that has been attached to the substrate, wherein additional copper (e.g., for interconnects) is added as needed (e.g., by electroplating). Then, it may be necessary to roughen the copper surface, for example, by etching, to allow the adhesion layer to be attached.

[0115] Thus, in some embodiments, a biochip (sometimes referred to herein as a "chip") is disclosed that includes a substrate that includes a plurality of electrodes (such as gold electrodes). The plurality of electrodes form an array. Each electrode may include a self-assembled monolayer as outlined herein and known to the skilled person. In an embodiment, one of the substances that form the monolayer comprises a capture ligand as outlined herein and known to the skilled person. In addition, each electrode has an interconnect that is attached to the electrode at one end and ultimately to a device that can control the electrode. That is, each electrode is independently addressable.

[0116] The substrate can be part of a larger device including a detection chamber that exposes a given volume of sample to the detection electrodes. Generally speaking, the detection chamber ranges from about 1 μl to 1 ml, or about 10 μl to 500 μl. As will be appreciated by those skilled in the art, smaller or larger volumes may be used depending on experimental conditions and assays.

[0117] In some embodiments, the detection chamber and electrodes are part of a cartridge that can be placed into a device that includes electronic components (AC / DC voltage source, ammeter, processor, readout display, temperature controller, light source, etc.) In this embodiment, the interconnects from each electrode are positioned so that when the cartridge is inserted into the device, a connection is established between the electrode and the electronic components.

[0118] The detection electrodes on the circuit board material (or other substrate) are usually prepared in a variety of ways. Generally speaking, high purity gold is used and can be deposited on the surface via a vacuum deposition process (sputtering and evaporation) or solution deposition (electroplating or chemical plating process). When the electroplating is completed, the substrate must initially contain a conductive material; fiberglass circuit boards are often provided with copper foil. Often, depending on the substrate, an adhesion layer is used between the substrate and the gold to ensure good mechanical stability. Therefore, some embodiments utilize a deposited layer of an adhesion metal (such as chromium, titanium, titanium / tungsten, tantalum, nickel or palladium) that can be deposited as above for gold. When using electroplated metal (adhesion metal or electrode metal), a grain refining additive (often referred to as a brightener in the industry) can be optionally added to change the surface deposition characteristics. Brighteners are mixtures of organic and inorganic substances (such as cobalt and nickel).

[0119] Generally speaking, the thickness of the adhesive layer is from about To about 25 microns (1000 micro inches). If the adhesion metal is electrochemically active, the electrode metal must be applied to a thickness that prevents "bleed-through"; if the adhesion metal is not electrochemically active, the electrode metal can be thinner. Generally speaking, the electrode metal (gold) is deposited to a thickness ranging from about To about 5 microns (200 micro inches), wherein the thickness is from about 30 micro inches to about 50 micro inches. Generally speaking, gold is deposited to form electrodes with a size range of from about 5 microns to about 5 mm in diameter, or from about 100 microns to 250 microns. The detection electrode thus formed is then cleaned and SAM is added as described below.

[0120] Therefore, a method for preparing a substrate comprising a plurality of gold electrodes is provided. The method first comprises applying an adhesion metal such as nickel or palladium (optionally with a brightener) to the substrate. Then the electrode metal (such as gold) is applied to the adhesion metal via electroplating. Then, a pattern of a device comprising electrodes and their associated interconnects is made using lithographic techniques (particularly photolithographic techniques as known in the art) and wet chemical etching. Often, these photolithographic techniques are used to lay down non-conductive chemically resistive insulating materials (such as solder masks or plastics), leaving only the electrodes and exposed connection points to the leads; the leads themselves are usually coated.

[0121] Self-assembled monolayer

[0122] This method can continue to add SAM (not in Figure 1a and Figure 1b ), which in some embodiments is presented via a capture probe adapter ( Figure 1a and Figure 1b One or more connectors of a similar form to the element 2a) in the electrode surface are also attached to the electrode surface, and are used to prevent or reduce undesirable electron transfer events to the electrode surface. In some embodiments, the required chemical substances, i.e., substances forming a monolayer, one of which is a capture ligand, are added using a drop deposition technique. As is well known, drop deposition techniques are used to prepare "dot" arrays. This is done to add different compositions to each electrode, i.e., to prepare arrays containing different capture ligands. Alternatively, the SAM material can be the same for each electrode, and this can be achieved using a drop deposition technique or by immersing the entire substrate or the surface of the substrate into a solution.

[0123] The present system is particularly useful in array format, i.e., a matrix in which addressable detection electrodes (generally referred to herein as "pads," "addresses," or "micropositions") are present. In this article, "array" means a plurality of capture ligands in array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 to thousands of different capture ligands can be made. In general, the array will contain from two to as many as 100,000 or more, depending on the size of the electrodes and the end use of the array. The range is from about 2 to about 10,000, about 5 to about 1000, and from about 10 to about 100. In some embodiments, the composition may not be in array form; that is, for some embodiments, a composition containing a single capture ligand may also be made. In addition, in some arrays, multiple substrates with different or identical compositions may be used. Thus, for example, a large array may include multiple smaller substrates.

[0124] In an embodiment, although this is not required in many systems, the electrode includes a self-assembled monolayer (SAM). As outlined herein, when the analyte is at a distance from the electrode, the efficiency of the target analyte binding (e.g., oligonucleotide hybridization) can be increased. Similarly, when there is a monolayer, the non-specific binding of biomolecules (including target analytes) to the electrode is generally reduced. Therefore, the monolayer is conducive to keeping the analyte away from the electrode surface. In addition, the monolayer is used to keep the charged substance away from the surface of the electrode. Therefore, this layer helps prevent electrical contact between the electrode and the ETM or between the charged substance in the electrode and the solvent. Such contact can cause a direct "short circuit" or an indirect short circuit caused by a charged substance that may be present in the sample. Therefore, the monolayer is tightly stacked on the electrode surface in the form of a uniform layer so that there is a minimum "hole". Therefore, the monolayer acts as a physical barrier to prevent the solvent from contacting the electrode.

[0125] SAM can comprise only conductive oligomer or comprise the mixture of conductive oligomer and insulator.As outlined herein, the use of monolayer reduces the amount of non-specific binding of biomolecules to surface, and in the case of nucleic acid, increases the efficiency of oligonucleotide hybridization due to the distance of oligonucleotide from electrode.Therefore, monolayer is conducive to keeping target analyte away from electrode surface.In addition, monolayer is also used to keep charge carrier away from the surface of electrode.Therefore, this layer helps to prevent the electrical contact between electrode and electron transfer part (ETM; Redox activity) or between electrode and charged substance in solvent.Such contact can cause direct "short circuit" or indirect short circuit caused by charged substance that can be present in sample.Therefore, monolayer is closely stacked on electrode surface in the form of uniform layer, so that there is minimum "hole".Therefore, monolayer can be used as physical barrier to prevent solvent and undesirable signal ("noise") from contacting electrode.

[0126] Using disposable devices for nucleic acid testing

[0127] An exemplary process is generally described. Although various elements (steps) are discussed as sequential steps with a specified order, it should be understood that the process is exemplary and is not intended to be limiting. One of ordinary skill will recognize that many of the various elements (steps) can be performed in an order different from the order described herein, can be performed simultaneously or substantially simultaneously with other elements (steps), or can be omitted entirely. Therefore, the order of the elements (steps) discussed is not limiting.

[0128] Step 1: Load the sample.

[0129] Step 2: Extracting nucleic acids, such as, for example, DNA.

[0130] Step 3: Combining nucleic acid (such as, for example, DNA) with amplification reagents comprising an ETM-labeled primer (ie, a signal primer).

[0131] Step 4: Amplify nucleic acid (such as, for example, DNA) to produce double-stranded signal amplicons.

[0132] Step 5: Incubate the double-stranded signal amplifier with exonuclease to form a single-stranded signal amplifier.

[0133] Step 6: Combine the single-stranded signal amplifier with the capture probe.

[0134] Step 7: Electrical sensor detection.

[0135] In some embodiments, the process in step 2 may include extracting RNA and may further include step 3a, reverse transcribing the RNA to provide cDNA.

[0136] General PCR

[0137] The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template in vitro to produce a specific DNA fragment. A typical amplification reaction comprises a target DNA, a thermostable polymerase (such as, for example, DNA polymerase), two oligonucleotide primers (5' and 3'), deoxynucleotide triphosphates (dNTPs), a reaction buffer, and a divalent cation (such as, for example, Mg). 2+ and / or Mn 2+). Each cycle of PCR includes the steps of template denaturation, primer annealing and primer extension. The initial step denatures the target DNA by heating it. During the denaturation process, the two intertwined DNA chains separate from each other, thereby generating the necessary single-stranded DNA template to be replicated by a thermostable DNA polymerase. In the next step of the cycle, the temperature is reduced so that the oligonucleotide primers can form a stable association (annealing) with the denatured target DNA and act as primers for the DNA polymerase. Finally, the synthesis of new DNA begins. The enzyme known as "Taq polymerase" uses the original chain as a template to synthesize ("build") two new DNA chains. This process causes the replication of the original DNA, wherein each of the new molecules contains an old and a new DNA chain. Then, each of these chains can be used to form two new copies, and so on. The cycle of denaturation and synthesis of new DNA is repeated up to 30 or 40 times, thereby generating more than one billion accurate copies of the original DNA fragment. The cyclic process of PCR is usually automated in a thermal cycler, which is programmed to change the temperature of the reaction to allow DNA denaturation and synthesis.

[0138] There are several nucleic acid amplification techniques that differ from traditional PCR reactions in that the reaction is performed at a single temperature. These isothermal methods include cyclic probe reactions, strand displacement, Invader TM , SNPase, rolling circle reaction and NASBA. The methods and systems described herein can also be used with isothermal methods.

[0139] In some embodiments, in the absence of nucleic acid synthesis, the primer cannot bind to the capture probe, and there should be little or no electron transfer between the signal primer and the electrode. In other embodiments, in the absence of nucleic acid synthesis, the primer can bind to the capture probe, but the electron transfer is undetectable or below a predetermined limit.

[0140] The selection of suitable PCR conditions is within the purview of those of ordinary skill in the art. Those skilled in the art will appreciate that it may be necessary to adjust the concentration of nucleic acid targets, primers, and the temperature of each step in order to optimize the PCR reaction for a given target and primers. Such optimization does not require excessive experimentation.

[0141] Types of PCR

[0142] The signal primers disclosed herein can be used in any amplification reaction, including PCR, 5-RACE, anchored PCR, "single-sided PCR", LCR, NASBA, SDA, RT-PCR, real-time PCR, quantitative PCR, quantitative RT-PCR, and other amplification systems known in the art (including universal primer formats).

[0143] In some embodiments, the signal primer has two regions: a region that is capable of binding to the DNA template during PCR amplification (e.g. Figure 2 a) as described in item "i" above, and a second region capable of generating a signal during redox-mediated electron detection (e.g. Figure 2 a as described in item "ii"). In some embodiments, the signal primer comprises three regions: a region capable of binding to a DNA template during PCR amplification ( Figure 2 l item "i"), a second region capable of generating a signal during redox-mediated electron detection ( Figure 2 l item "ii"), and a third area connecting the first area and the second area ( Figure 2 l, joints symbolized by a triangle).

[0144] The sequence amplified by the signal primer is selected based on the known target sequence so that if hybridization to the complementary target sequence occurs, amplification proceeds at a significant and detectable rate.

[0145] PCR reactions can involve dramatic temperature fluctuations. In this article, it is not known whether the signal primer can withstand these temperature fluctuations. For example, high temperatures (e.g., 90°C to 99°C) for denaturing double-stranded DNA, lower temperatures (e.g., 40°C to 60°C) at which DNA primers are hybridized to DNA templates, and hybridization temperatures (e.g., 60°C to 80°C).

[0146] Assay development

[0147] When developing assays utilizing prior art sandwich assays (capture probe combined with an amplicon bound to a signal probe), it can be difficult to find a portion of a target organism sequence that is highly conserved (i.e., detectable across many variants) but also uniquely identifies the target organism of interest. This is even more complicated when the highly conserved yet unique identifier must be long enough to allow both capture and signal probe binding.

[0148] A key advantage of the provided method and system is that there is no need to develop a signal probe. In other words, there is no need to develop an amplicon with a signal probe binding region. Therefore, the amplicon produced can be much shorter, making the assay design simpler and faster. This can result in shorter extension times, sharper melting temperatures, and overall higher efficiency in each round of amplification, because the amount of synthesis is reduced compared to traditional sandwich assay systems. This can also result in a higher detection signal, and therefore a higher sensitivity.

[0149] Described is an assay that does not utilize a signaling probe.Described is an assay that does not utilize a sandwich assay during detection.

[0150] Because the unbound signal primer is driven away from the electrode when a voltage is applied, there is no need to separate the signal primer from the single-stranded signal amplicon. Unlike fluorescence-based systems that require the use of quenchers, the present system does not require the use of such donor / quencher dye pairs.

[0151] Design of synthetic oligonucleotides

[0152] Regarding the design of synthetic oligonucleotides for use in amplification reactions, Rychlik et al. (1989, Nucleic Acids Research, Vol. 17 (21): 8543-8551) and Rychlik (1995, Molecular Biotechnology, Vol. 3: 129-134) describe selection criteria and computer programs for designing probes and primers (including primers for in vitro amplification of DNA). Both teach that primers should not generate secondary structures or exhibit self-hybridization. U.S. Pat. No. 6,495,323 (the entire contents of which are incorporated by reference) describes in detail the formation of probes attached to electron transfer moieties. The same process used to prepare ferrocene-labeled probes can be used to prepare ferrocene-labeled primers.

[0153] General probe synthesis, functionalization, and conjugation

[0154] US Patent No. 10,001,476 discloses probe synthesis, functionalization, and conjugation in greater detail, and the entire disclosure of which is incorporated by reference.Probe synthesis, functionalization, and conjugation are all techniques well known in the art.

[0155] Nucleic acid capture probes are typically designed to be complementary to a sequence of approximately 40 to 50 bases within the target nucleic acid. The capture probe sequence is typically complementary to the 3'-region of the target nucleic acid (but vice versa, i.e., the 5'-region of the target nucleic acid may also be complementary) and is designed to have a melting temperature (T) of approximately 50°C. M ). The capture probe can be modified at the 3' end or the 5' end with a disulfide linker for covalent attachment to the gold electrode surface, for example, essentially as described in commonly owned US Pat. No. 6,753,143 and US Pat. No. 7,820,391, each of which is incorporated herein by reference in its entirety.

[0156] Capture probes (including, for example, nucleic acids) can be attached directly or indirectly, covalently or non-covalently to electrodes or other substrate surfaces using a variety of well-known techniques. See, for example, Chapter 13, Chemically Modified Electrodes, Martin and Foss, pages 403 to 442, Laboratory Techniques in Electroanalytical Chemistry; 2nd edition, Kissinger and Heineman, ed., MARCEL DEKKER, INC. (1996); Biochip Technology, Cheng and Kricka, ed., George H. Buchanan Printing Company, Bridgeport, NJ (2001). In some embodiments, this is accomplished by mixing a disulfide self-assembled monolayer insulator sequence precursor with a 3' or 5' modified nucleic acid capture probe with a disulfide group as described above, and spotting onto a gold or gold-plated electrode. This is mediated by a linker / functional group, e.g., W330, as cited and described in U.S. Pat. No. 7,820,391 (incorporated by reference in its entirety), or N150, as cited and described in U.S. Pat. No. 6,753,143 (incorporated by reference in its entirety). As the skilled person will appreciate, there are many types of linkers available, e.g., as described in the previously cited documents.

[0157] Signal primer synthesis

[0158] The signal primer sequence is complementary to a specific region of the target. If sequence differentiation is required, the sequence polymorphism should be as close to the center of the amplicon sequence as possible, and the T of the two amplicons should be M Should match as closely as possible.

[0159] When preparing a signal primer, the electron transfer moiety (ETM) can be covalently attached to a variety of locations on the nucleic acid: the 5'-end ( Figure 2 c) the middle of the sequence ( Figure 2 b) or in both Figure 2 a). In embodiments, the attachment is via attachment to the base of the nucleoside or via attachment to the backbone of the nucleic acid, including attachment to the ribose in the ribose-phosphate backbone or to the phosphate moiety. In embodiments, the composition is designed so that the electron transfer moiety is as close to the "π-way" as possible.

[0160] In some embodiments, the signal primer is designed with a ferrocene label (such as, for example, N6, QW56 or QW80). The attachment of the ferrocene label should not disrupt the Watson-Crick base pairing of the primer to which the electron transfer moiety is attached, nor disrupt the annealing of the primer to the target. In other embodiments, the primer may contain an additional terminal nucleoside (n+1 or n+2) at the end of the nucleic acid, which is used to covalently attach the electron transfer moiety (ETM) but which does not participate in base pair hybridization, similar to Figure 2 j and Figure 2 In yet other embodiments, it may be desirable to insert a linker arm that separates the electron transfer moiety from the primer region, such as Figure 2 l and Figure 2 In yet other embodiments, it may be desirable to have more than one signal portion on the primer region, such as Figure 2 n. In some embodiments, one ferrocene marker is added to the signal primer sequence. In some embodiments, two ferrocene markers are added to the signal primer sequence. In some embodiments, three ferrocene markers are added to the signal primer sequence. In some embodiments, four ferrocene markers are added to the signal primer sequence. In some embodiments, five ferrocene markers are added to the signal primer sequence. In some embodiments, six ferrocene markers are added to the signal primer sequence. In some embodiments, seven ferrocene markers are added to the signal primer sequence. In some embodiments, eight ferrocene markers are added to the signal primer sequence. In some embodiments, nine ferrocene markers are added to the signal primer sequence. In some embodiments, ten ferrocene markers are added to the signal primer sequence. In some embodiments, one to ten ferrocene markers are added to the signal primer sequence. In some embodiments, one to six ferrocene markers are added to the signal primer sequence. In some embodiments, six ferrocene markers are added to each signal primer.

[0161] In some embodiments, the signal primer is designed to have a ferrocene label (such as, for example, N6, QW56 or QW80) at or near the 5'-end of the non-phosphorylated oligonucleotide signal primer (e.g., attached to any one of nucleotides 1 to 3 at the 5'-end of the signal, or attached to any one of nucleotides 1 to 2 at the 5'-end). In some embodiments, a ferrocene label is added to the 5'-end of the signal primer sequence. In some embodiments, one ferrocene label is added to the 5'-end of the signal primer sequence. In some embodiments, two ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, three ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, four ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, five ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, six ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, seven ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, eight ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, nine ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, ten ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, one to ten ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, one to six ferrocene labels are added to the 5'-end of the signal primer sequence. In some embodiments, six ferrocene labels are added to each signal primer. Because all hybridization reactions must be performed at a single temperature, the T of all signal primers is M Values ​​should all be within 5° C. Because all detection reactions must occur in the same solution, signal primers and capture probes must be designed to avoid any cross-hybridization; a maximum ΔG0 value for cross-hybridization has been established empirically.

[0162] Signal primers are synthesized using standard phosphoramidite chemistry and may contain any nucleotide or modified base suitable for DNA polymerase. The nucleic acid portion of the signal primer may be DNA or RNA or a chimeric mixture or derivative or modified form thereof. In addition to being labeled with an electrochemically detectable label, the signal primer may also be modified on the base portion, sugar portion, or phosphate backbone, and may include other additional groups or labels.

[0163] The signal primer can be any suitable size, but ideally does not bind to the capture probe without extension. In some embodiments, the signal primer is in the range of 10 to 100 nucleotides, or 10 to 80 nucleotides, or 11 to 40 nucleotides, or 17 to 25 nucleotides, although the signal primer can be longer or shorter as desired.

[0164] The signaling moiety of the signal primer may include at least one or more electron transfer moieties selected from the group including but not limited to ferrocene and ferrocene derivatives. In one embodiment, the signaling moiety of the signal primer may include one to six electron transfer moieties. In one embodiment, the signaling moiety of the signal primer may include one to ten electron transfer moieties.

[0165] In certain embodiments, the signal primer or the signal amplifier of single strand or the signal amplifier of double strand may contain one or more labels (which may be the same or different). On the one hand, the oligonucleotide is labeled and labeled as any part, which undergoes detectable electrochemical changes when hybridized with the capture probe on the electrode surface. In an embodiment, it is labeled as a ferrocene tag and the label undergoes detectable changes in one or more electrochemical properties. Such properties include but are not limited to signal intensity, electrochemical potential or reaction constant.

[0166] The signaling portion of the signal primer may be located at one or more positions within the signal primer and / or at or near the 5'-end. In one embodiment, the signaling portion of the signal primer may be located at the first or second 3'- or 5'-most nucleotide, the first or second or third 3'- or 5'-most nucleotide, one of the first five 3'- or 5'-most nucleotides, one of the first ten 3'- or 5'-most nucleotides, one of the first fifteen 3'- or 5'-most nucleotides, or one of the first twenty 3'- or 5'-most nucleotides. In a specific embodiment, the signaling portion of the signal primer is located at the first base at the 3'- or 5'-end.

[0167] Signal primers lack an enhancing group. They do not undergo a detectable change in any observable property upon hybridization and / or extension. The signaling portion of a signal primer is not a fluorescent portion.

[0168] Any known labeling method can be used to label the signal primer (as described above). For example, the signal primer can be labeled by: (1) attachment at the sulfur in the phosphorothioate bond; (2) attachment at the 2'-amino group; (3) attachment at the 1' position using, for example, a suitably modified sugar containing a carboxamide substituted with an alkylamine; (4) attachment at the 1' position using, for example, an abasic site and, for example, an alkyl diamine as a linker; (5) formation of a structure by reductive alkylation of an adduct formed between an alkyl diamine and an abasic site; (6) incorporation using 4'-thio-2'-deoxyuridine or 4'-thiothymidine; (7) attachment at 4-thiothymidine or 4-thio-2'-deoxyuridine. (8) at the 2'-position of oxyuridine; (9) at the 4-amino position of deoxycytidine if the 4-amino group is derivatized with an alkylamine; (10) using the 8' position of adenine for incorporation if this position is substituted with an alkylthioamine; (11) at the N2 position of guanine if the N2 amino group is derivatized with an alkylamino group; or (12) at the N2 position of aminoadenine if the 2-amino group is derivatized with an alkylamine.

[0169] All signal primers can be purified using techniques known in the art.

[0170] False positive results

[0171] False-positive results can lead to serious problems, including unnecessary use of antibiotic, antimicrobial, or antifungal therapy. To avoid false-positive results, the signal primer should not bind to the capture probe. For example, Figure 6 In the method, segment c is the extension part of the amplicon, segment b is the primer region, and segment a is the signal transduction part. Segment b cannot bind to the capture probe, thereby reducing false positives. In addition, in the method steps, a separation step can be used to separate the signal primer from the single-stranded signal amplicon.

[0172] False negative results

[0173] False negative results are rare. In prior art systems, the amplicon is bound to the signal probe. If there is an error in the function or design of the signal probe, a false negative will result. By eliminating the requirement for the amplicon to be bound to the signal probe, false negatives can be reduced.

[0174] Signal Primer

[0175] Unlike the capture probes used in the system, the signal primers do not bind to the electrodes prior to detection. During detection, they do not bind directly to the electrodes but to the capture probes, which hold them in place for detection.

[0176] Unlike signal probes used in prior art systems, signal primers participate in both amplification and detection.

[0177] In some embodiments, the primer is made of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, hexapolyethylene glycol-modified nucleic acid, chimeric mixtures or derivatives or modified versions thereof.

[0178] In some embodiments, the signal primer does not overlap with the capture probe. In some embodiments, the signal primer overlaps with the capture probe by about 1 to 10 base pairs. In some embodiments, the signal primer overlaps with the capture probe by about 1 to 12 base pairs.

[0179] The signal primer disclosed herein can be better understood by the following examples. In one embodiment, the signal primer comprises a nucleic acid and a detectable label. In some embodiments, the signal primer comprises a nucleic acid and an electron transfer portion. In certain embodiments, the signal primer comprises a nucleic acid and a ferrocene label. In another embodiment, the signal primer comprises a nucleic acid and an electron transfer portion, wherein the electron transfer portion is attached to the 5'-end of the primer. In some embodiments, the signal primer comprises a nucleic acid and a label capable of generating a signal during redox-mediated electron detection. In some embodiments, the signal primer comprises a nucleic acid and an electrochemically detectable label. In some embodiments, the signal primer comprises 5 to 12 nucleic acids and an electrochemically detectable label. In some embodiments, the signal primer comprises 5 to 25 nucleic acids and an electrochemically detectable label. In some embodiments, the signal primer is a self-annealing primer and comprises an electrochemically detectable label. In some embodiments, the signal primer comprises a nucleic acid and an electron transfer portion, wherein the electron transfer portion is attached to the 5'-end of the primer and can generate a signal during redox-mediated electron detection. In some embodiments, the signal primer comprises a nucleic acid and an electrochemically detectable label, wherein the electrochemically detectable label is attached to the 5'-end of the primer. In some embodiments, the signal primer consists of a nucleic acid and an electrochemically detectable label.

[0180] Determine target concentration

[0181] When electrochemical detection is used, the primary goal of the nucleic acid amplification step is to generate detectable nucleic acids from the target molecule at a concentration of about 0.01 pmolar, as this has been found to be within the lower limit of detection for electrochemical detection. 3 DNA molecules, then one milliliter (a reasonably accessible sample volume) contains 5×106 molecules or about 10 7 To increase the amount of DNA in 1 ml of blood to 0.01 pmol of DNA requires about 10 3 This can of course be achieved using several well-known amplification techniques. It will be apparent to those skilled in the art that similar calculations will be performed for different sample types and sample volumes to determine the extent of amplification.

[0182] Primer removal after amplification

[0183] It has been found that the results of seeking to develop systems that incorporate rapid PCR reactions (i.e., amplification that is completed in less than about 10 minutes) are necessary to increase the primer concentration. As described above, if there is an excess of primer in the mixture during detection, this may generate a "primer background" in the detection step that may interfere with or reduce the signal generation of the single-stranded signal amplicon bound to the capture oligonucleotide.

[0184] Co-owned U.S. Patent No. 10,864,522 (the entire contents of which are incorporated herein by reference) describes a processing box and a method for detecting pathogens in a sample. Specifically, the 10,864,522 patent describes moving samples, primers and amplifiers on a digital microfluidic path. In such systems, primers and amplifiers will have different charge-to-mass ratios so that they can be separated from each other by moving them along the digital microfluidic path. The material of PCR double-stranded amplification is moved by the digital microfluidic path, wherein the smaller synthetic oligonucleotide primer moves the fastest, and the larger amplifier moves slower. After a certain amount of time, the material amplified by PCR can be separated on the digital microfluidic path, wherein half is an oligonucleotide primer, and the other half is a larger amplifier. In certain embodiments, the separation occurs after the material amplified by PCR has been denatured with an endonuclease. In one embodiment, a membrane or gel can be added to the digital microfluidic path, which slows down the speed of the macromolecule compared to short molecules to help separate the material.

[0185] Detectable Label

[0186] Detectable label can include electron transfer part.Electron transfer part includes ferrocene label.In some embodiments, ferrocene label (such as, for example, N6, QW56 or QW80) is on the 5'-end of primer or near the 5'-end of primer (for example, attached to any one of nucleotides 1 to 3 at 5'-end, or attached to any one of nucleotides 1 to 2 at 5'-end).However, ferrocene label can also be on the 3'-end of primer or on both 5'- and 3'-end.Signal primer produces double-stranded signal amplifier.When double-stranded signal amplifier denatures, it forms single-stranded signal amplifier, which can bind capture probe and generate electrochemical signal.Therefore, signal primer plays a role in both amplification and detection.Signal primer allows system to utilize shorter signal amplifier.In some embodiments, amplifier is about 60 base pairs.In some embodiments, amplifier is about 30 to 120 base pairs.In some embodiments, amplifier is about 70 to 250 base pairs. In some embodiments, the amplicon is about 50 to 500 base pairs.

[0187] In one embodiment, the nucleic acid is modified with at least one electron transfer moiety at one position. In one embodiment, the nucleic acid is modified with at least two electron transfer moieties at two positions (see Figure 2 n). In one embodiment, the nucleic acid is modified with more than two electron transfer moieties at more than two positions. In one embodiment, the nucleic acid is modified with multiple electron transfer moieties at multiple positions. For example, to increase the signal obtained from the primer, multiple electron transfer moieties may be used at multiple positions. For example, the electron transfer moieties may be attached at both the 5' and 3' positions, such as at Figure 2 n. In one embodiment, the plurality of electron transfer moieties are identical to produce a uniform signal. In another embodiment, each of the plurality of electron transfer moieties can be different. In one embodiment, the two electron transfer moieties are identical to produce a uniform signal. In another embodiment, the two electron transfer moieties can be different.

[0188] In certain embodiments, detectable label is maintained as far away from capture probe one base pair and still produces detectable signal.In certain embodiments, detectable label is maintained at any position far away from capture probe 1 to 10 base pairs and still produces detectable signal.In certain embodiments, detectable label is maintained at any position far away from capture probe 1 to 50 base pairs and still produces detectable signal.In certain embodiments, detectable label is maintained at any position far away from capture probe 1 to 100 base pairs and still produces detectable signal.In certain embodiments, detectable label is maintained at any position far away from capture probe 36 to 72 base pairs and still produces detectable signal.Therefore, capture probe signal primer complex is provided herein, wherein ETM (such as, for example, ferrocene label) is far away from capture probe and its hybridization position from 1 to 10 base pairs.In certain embodiments, capture probe signal primer complex is provided, wherein ETM (such as, for example, ferrocene label) is far away from capture probe and its hybridization position from 1 to 50 base pairs. In a further embodiment, provided herein is a capture probe signal primer complex, wherein the ETM (such as, for example, a ferrocene label) is from 1 to 100 base pairs away from the position to which the capture probe hybridizes. In a further embodiment, provided herein is a capture probe signal primer complex, wherein the ETM (such as, for example, a ferrocene label) is from 36 to 72 base pairs away from the position to which the capture probe hybridizes.

[0189] Single-stranded signal amplicon

[0190] Single-stranded signal amplicon Figure 2 (d to f), where "ii" is a detection moiety (e.g., ferrocene) and "i" is an annealing region (e.g., a region that binds to a capture probe during detection). In some embodiments, the amplicon comprises two regions: one region that is capable of binding to a capture probe, and a second region that is capable of generating a signal during redox-mediated electronic detection. In some embodiments, the amplicon comprises three regions: one region that is capable of binding to a capture probe, a second region that is capable of generating a signal during redox-mediated electronic detection, and a third region that connects the first region and the second region.

[0191] In the presence of an appropriate target and a DNA polymerase with associated components, the disclosed synthetically labeled oligonucleotide primers are used to perform PCR to generate newly synthesized DNA molecules with an incorporated signal moiety region. The molecule can then be denatured and hybridized to the target sequence on a solid support. The signal moiety region can then be used to generate a signal. In some embodiments, the signal moiety is a ferrocene label.

[0192] In some embodiments, the length of the single-stranded signal amplifier is 60 bases. In some embodiments, the length of the single-stranded signal amplifier is 30 to 60 bases. In some embodiments, the length of the single-stranded signal amplifier is 20 to 100 bases. In some embodiments, the length of the single-stranded signal amplifier is 20 to 150 bases.

[0193] In some embodiments, the amplicon is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, and hexapolyethylene glycol-modified nucleic acid.

[0194] In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and a detectable label. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and a ferrocene label. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, and the nucleic acid comprises a first portion and a second portion, wherein the first portion and the second portion are different.

[0195] In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, the nucleic acid comprising a first portion and a second portion, wherein the first portion and the second portion are different, and wherein the first portion is capable of binding to a first capture probe and the second portion is capable of binding to a second capture probe. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, the nucleic acid comprising a first portion and a second portion, wherein the first portion and the second portion are different, and wherein the first portion is capable of binding to a first capture probe but not to a second capture probe, and the second portion is capable of binding to a second capture probe but not to a first capture probe. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, the nucleic acid comprising a first portion and a second portion, wherein the first portion and the second portion are different, and wherein the first portion is capable of binding to a first capture probe and a second capture probe. In some embodiments, the single-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, the nucleic acid comprising a first portion and a second portion, wherein the first portion and the second portion are different, and wherein the first portion is capable of binding to a first capture probe and a second capture probe, and wherein the second portion is capable of binding to a first capture probe and a second capture probe. In certain embodiments, the electron transfer part is attached to the 5'-end of the signal amplifier of the single chain or the 5'-end of the signal amplifier of the single chain (for example, attached to any one of the nucleotides 1 to 3 at the 5'-end or attached to any one of the nucleotides 1 to 2 at the 5'-end). In certain embodiments, the signal amplifier of the single chain comprises an electrochemically detectable label. In certain embodiments, the signal amplifier of the single chain comprises a capture probe binding region, a primer binding region, and an electrochemically detectable label region. In this article, in certain embodiments, the electrochemically detectable label region is 5 to 100 base pairs away from the capture probe binding region. In certain embodiments, the electron transfer part is an electrochemically detectable label. In this article, in certain embodiments, the electron transfer part is an electrochemically detectable label and is attached to the 5'-end of the signal amplifier of the single chain or the 5'-end of the signal amplifier of the single chain. In certain embodiments, the signal amplifier of the single chain consists of a capture probe binding region, a primer binding region, and an electrochemically detectable label region. In this article, in certain embodiments, the electrochemically detectable label is 5 to 100 base pairs away from the capture probe binding region. In some embodiments, the single-stranded signal amplifier comprises an electrochemically detectable label attached to the 5'-end and the 3'-end of the single-stranded signal amplifier.

[0196] Double-stranded amplicon

[0197] In some embodiments, the length of the double-stranded signal amplifier is 60 base pairs. In some embodiments, the length of the double-stranded signal amplifier is 30 to 60 base pairs. In some embodiments, the length of the double-stranded signal amplifier is 20 to 100 base pairs. In some embodiments, the length of the double-stranded signal amplifier is 20 to 150 base pairs.

[0198] In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and a detectable label. In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and an electron transfer portion. In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and a ferrocene label. In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, and the nucleic acid comprises a first portion and a second portion, wherein the first portion and the second portion are different. In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, wherein the electron transfer portion is attached to the 5'-end of the double-stranded signal amplifier or near the 5'-end of the double-stranded signal amplifier. In some embodiments, the double-stranded signal amplifier comprises a nucleic acid and an electron transfer portion, wherein the electron transfer portion is attached to the forward end and the reverse end of the double-stranded signal amplifier. In some embodiments, the double-stranded signal amplifier comprises an electrochemically detectable label.

[0199] Hybridization complex: capture probe and signal amplifier

[0200] In some embodiments, the forward primer and reverse primer are labeled with an electrochemical detectable label. In some embodiments, the forward primer is labeled with an electrochemical detectable label. In some embodiments, the reverse primer is labeled with an electrochemical detectable label. In some embodiments, there is a first capture probe specific to the forward primer labeled with the electrochemical detectable label and a second capture probe specific to the reverse primer labeled with the electrochemical detectable label.

[0201] In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and a detectable label. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and an electron transfer portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and a ferrocene label. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and an electron transfer portion, wherein the nucleic acid comprises a first portion and a second portion, wherein the first portion and the second portion are different. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and an electron transfer portion, wherein the nucleic acid comprises a first portion and a second portion, and the capture probe is capable of binding to the first portion but not to the second portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and an electron transfer portion, wherein the nucleic acid comprises a first portion and a second portion, and the capture probe is capable of binding to the second portion but not to the first portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier, the amplifier comprising a nucleic acid and an electron transfer portion, wherein the nucleic acid comprises a first portion and a second portion, and the capture probe is capable of binding to the second portion and the first portion. In some embodiments, the hybridization complex comprises a capture probe bound to an amplifier comprising a nucleic acid and an electron transfer moiety, wherein the hybridization complex is bound to an electrode, the electrode comprising a monolayer.

[0202] method

[0203] The signal primers disclosed herein can be used in diagnostic methods, wherein the signal primer is complementary to the sequence (e.g., genome or cDNA) of an infectious disease pathogen, or can initiate the synthesis or amplification of the sequence of an infectious disease pathogen (e.g., human disease) (including but not limited to viruses (e.g., HIV, HPV, etc.), bacteria, parasites and fungi), thereby diagnosing the presence of infectious pathogens in samples from patients. The type of target nucleic acid can be genomic, cDNA, mRNA or synthetic, or the source can be human, animal, fungus or bacteria. In another embodiment that can be used for the diagnosis or prognosis of a disease or illness, the target sequence is a wild-type human genomic DNA or RNA, or cDNA sequence whose mutation is involved in the presence of a human disease or illness, or alternatively can be a mutated sequence. In such embodiments, different sets of signal primers (e.g., signal primers labeled in different ways) that selectively identify wild-type sequences or mutant forms can be used to repeat the amplification reaction for the same sample. For example, the mutation can be an insertion, substitution and / or deletion, or a translocation of one or more nucleotides. In another embodiment, the signal primer can be used in SNP analysis, pharmacogenomics and toxicogenetics.

[0204] In a specific embodiment, a method for detecting or measuring the products of a nucleic acid amplification or synthesis reaction is disclosed, which comprises: (a) contacting a sample containing or suspected of containing one or more target nucleic acid molecules with one or more signal primers (such primers comprise one or more labels, which may be the same or different and may be labeled internally and / or at or near the 3'-end and / or at or near the 5'-end), wherein the primers are suitable for use in the amplification or synthesis reaction such that the primers are incorporated into the amplified or synthesized products of the amplification or synthesis reaction when the target sequence or nucleic acid molecule is present in the sample; (b) performing an amplification or synthesis reaction; and (c) detecting or measuring one or more synthesized or amplified product molecules by redox-mediated electronic detection.

[0205] In another embodiment, a method for detecting a target nucleic acid sequence is disclosed, comprising contacting a sample containing a mixture of nucleic acids with at least one oligonucleotide capable of hybridizing with the target nucleic acid sequence and comprising at least one detectable portion, wherein the detectable portion undergoes a redox reaction after an electrical signal is applied thereto, wherein a change in redox potential indicates the presence of the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is not separated from the mixture.

[0206] In another embodiment, a method for quantifying a target nucleic acid molecule is disclosed, comprising contacting a sample containing a mixture of nucleic acids comprising the target nucleic acid molecule with at least one oligonucleotide containing a detectable moiety, wherein the detectable moiety undergoes a detectable redox reaction upon application of a charge and observing the reaction, wherein the observable reaction is proportional to the amount of the target nucleic acid molecule in the sample.

[0207] In another embodiment, a method for amplifying one or more nucleic acid molecules is disclosed, comprising (a) mixing one or more templates or target nucleic acid molecules with one or more signal primers; and (b) incubating the mixture under conditions sufficient to synthesize or amplify one or more nucleic acid molecules complementary to all or a portion of the template or target molecule. In one embodiment, the amplified nucleic acid molecules include one or more signal primers. On the one hand, the signal primer is incorporated at or near one or both ends of the synthetic or amplified nucleic acid molecules produced. One or more nucleic acid molecules produced by such amplification or synthesis reaction are also disclosed.

[0208] In another embodiment, a method for determining the presence or absence of a target in a sample is disclosed. In some embodiments, the target sequence is a wild-type human genome or RNA or cDNA sequence. In some embodiments, the target sequence is a mutated human genome or RNA or cDNA sequence. The mutation is involved in the presence of a human disease or illness. In some embodiments, a signal primer amplifies a wild-type target, and in other embodiments amplifies a mutated target. For example, a mutation can be an insertion, substitution and / or deletion of one or more nucleotides, or a translocation. In another embodiment, a signal primer can be used in SNP analysis, pharmacogenomics, and toxicogenetics.

[0209] In a specific embodiment, a method for detecting the presence of nucleic acid amplification products is disclosed, which comprises: (a) contacting a sample containing or suspected of containing one or more target nucleic acid molecules with one or more signal primers (such primers may contain one or more labels, which may be the same or different and may be labeled internally and / or at or near the 3'-end and / or at or near the 5'-end), wherein the signal primer is suitable for use in the amplification or synthesis reaction such that the signal primer is incorporated into the amplified product of the amplification reaction when the target sequence or nucleic acid molecule is present in the sample; (b) performing an amplification reaction; and (c) detecting or measuring one or more amplification product molecules by electrochemical detection.

[0210] In another embodiment, a method for determining the absence of at least one specific target or template nucleic acid molecule in a sample is disclosed, comprising: (a) contacting the sample with a signal primer, wherein at least a portion of the signal primer is capable of forming a base pair (e.g., hybridizing) with at least a portion of the target nucleic acid molecule; and (b) incubating the signal primer and nucleic acid molecule mixture under conditions sufficient to amplify the at least a portion of the target nucleic acid molecule. Under such conditions, the lack of amplification indicates the absence of one or more specific nucleotides in the sample.

[0211] In one embodiment, a method for performing a nucleic acid amplification assay is provided, comprising (a) combining reagents for a polymerase chain reaction, a polymerase (e.g., a DNA polymerase), a target nucleic acid, and a modified primer, wherein the modified primer comprises a detectable label; (b) circulating the mixture of (a) to provide multiple copies of the amplicon incorporated into the modified primer; (c) exposing the mixture in (b) to an exonuclease to produce a single-stranded amplicon; (d) exposing the mixture in (c) to a capture oligonucleotide complementary to the single-stranded signal amplicon incorporated into the modified primer; (e) hybridizing the single-stranded signal amplicon incorporated into the modified primer with the capture oligonucleotide; and (f) detecting the label associated with the hybridization. In some embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and their modifications and derivatives. In some embodiments, the target nucleic acid is extracted from blood, oral swabs, tissues, body fluids, environmental samples, material surfaces, plants, animals, bacteria, or fungi. In some embodiments, the polymerase is a DNA polymerase and is a Taq polymerase or a thermococcus kodakiensis polymerase. In some embodiments, the primer is selected from the following groups: deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, and hexapolyethylene glycol-modified nucleic acid. In some embodiments, the detectable label is an electron transfer moiety. In some embodiments, the detectable label is a ferrocene label. In certain embodiments, the detectable label is N6, QW56, or QW80. In some embodiments, the cycle is isothermal. In some embodiments, the cycle is between the first temperature and the second temperature. In some embodiments, the capture oligonucleotide is fixed on a gold surface. In some embodiments, the capture oligonucleotide is fixed on an electrode surface. In some embodiments, the detection is electrochemical. In some embodiments, the label is not exposed to a second portion or quencher with a second label.

[0212] In another embodiment, a method for performing a nucleic acid amplification assay is provided, comprising (a) combining PCR reagents, a polymerase (e.g., a DNA polymerase), a target nucleic acid or a sample suspected of containing a target nucleic acid, a modified primer and a second unmodified primer, wherein the modified primer comprises a ferrocene label; (b) cycling the mixture in (a) to provide multiple copies of a first amplicon incorporated into the modified primer and a second amplicon incorporated into the second unmodified primer in the presence of the target nucleic acid in the sample; (c) exposing the mixture in (b) to a nuclease exonuclease to produce a first single-stranded signal amplicon incorporated into the modified primer and a second single-stranded amplicon incorporated into the unmodified primer; (d) exposing the mixture in (c) to a capture oligonucleotide complementary to the single-stranded signal amplicon incorporated into the modified primer; (e) hybridizing the single-stranded signal amplicon of the amplicon incorporated into the modified primer to the capture oligonucleotide; and (f) detecting the label associated with the modified primer incorporated into the amplicon. In some embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and their modifiers and derivatives. In some embodiments, the target nucleic acid is extracted from blood, oral swabs, tissues, body fluids, environmental samples, material surfaces, plants, animals, bacteria or fungi. In some embodiments, the reagent includes a reagent for polymerase chain reaction amplification. In some embodiments, the polymerase is a DNA polymerase and is a Taq polymerase. In some embodiments, the polymerase is a DNA polymerase and is a thermococcus kodakiensis polymerase. In some embodiments, the primer is selected from the following groups: deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid and hexapolyethylene glycol-modified nucleic acid. In some embodiments, the detectable label is selected from the group consisting of N6, QW56 and QW80. In some embodiments, the detectable label is ferrocene, methylene blue or osmium. In some embodiments, the cycle is isothermal. In some embodiments, the cycle is between the first temperature and the second temperature. In some embodiments, the capture oligonucleotide is fixed on a gold surface. In some embodiments, the capture oligonucleotide is fixed on an electrode. In some embodiments, a signal amplifier incorporating a single strand of a modified primer is hybridized to a capture oligonucleotide. In some embodiments, the detecting is electrochemical.

[0213] Integrated nucleic acid test kit

[0214] Integrated multiplex target analysis systems are known and described in US 10,864,522 (which is incorporated by reference).

[0215] Provided herein is an integrated nucleic acid test kit capable of performing amplification and detection. In general, the integrated nucleic acid test kit is capable of receiving a sample, extracting DNA, combining the DNA with an amplification reagent comprising an ETM-labeled primer, amplifying the DNA, incubating the signal amplifier with an exonuclease to form a single-stranded signal amplifier, combining the single-stranded signal amplifier with a capture probe, and performing electrical sensor detection.

[0216] In certain embodiments, the target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and their modifications and derivatives, and in other embodiments, the target nucleic acid is extracted from blood, oral swabs, tissues, body fluids, environmental samples, material surfaces, plants, animals, bacteria and fungi.

[0217] Further disclosed herein is an integrated nucleic acid test kit capable of performing amplification and detection. Further disclosed herein is an integrated nucleic acid test kit for use with electrochemical detection. Further disclosed herein is an integrated nucleic acid test kit capable of performing amplification. Further disclosed herein is an integrated nucleic acid test kit capable of performing detection. Further disclosed herein is an integrated nucleic acid test kit capable of performing amplification and detection. Further disclosed herein is an integrated kit for nucleic acid testing that operates in conjunction with a reader instrument.

[0218] Reagent test kit

[0219] Kits for detecting nucleic acid molecules in a sample are disclosed. Such kits can also be designed to detect target nucleic acid molecules during or after a nucleic acid amplification reaction. Such kits can be diagnostic kits, where the presence of nucleic acid is correlated with the presence or absence of a disease or disorder. Kits for performing amplification reactions described herein and kits for preparing compositions described herein are disclosed.

[0220] In certain embodiments, the kit comprises one or more signal primers as defined herein.The kit may further comprise additional components for performing a detection assay or other method.

[0221] Such kits may include one or more additional components selected from the group consisting of: one or more polymerases (e.g., DNA polymerases and reverse transcriptases), one or more nucleotides, one or more buffer salts (including nucleic acid amplification buffers), one or more control nucleic acid target molecules (for serving as positive controls for test assays), instructions for performing the methods, etc.

[0222] Examples

[0223] Example 1: Amplification and detection using signal primers

[0224] Amplification of SARS-CoV-2 amplicon by control primers or signal primers ( Fig.10 ). In this example, a reverse primer for the SARS-CoV2 N1 gene was synthesized with an N6 tag. A concurrent reverse primer without any tag was used as a control. After digestion with lambda exonuclease, the "control" amplicon was incubated with the SARS-CoV-2N1 signal probe. On the other hand, the amplicon generated by using the signal primer was digested with lambda exonuclease but not incubated with the signal probe. The amplicon was then loaded on the detection zone of the Respiratory Panel box of GenMark (Carlsbad, CA). The signal generated from the amplicon using the signal primer was higher than the signal generated using the control primer ( Fig.10 In addition, no false positive signals were detected from other targets in the detection zone, confirming that the signal primer did not bind nonspecifically to the capture probe ( Fig.11 ).

[0225] Example 2: Detection using a traditional sandwich assay (control primer) versus a signal primer (where ferrocene is far from the capture probe)

[0226] Adenovirus C, OC43, RSV A, and SARS-CoV-2 were amplified by a reverse primer without any tag present (“control primer”) or the corresponding signal primer. Fig.12 The distance between the ferrocene label and the capture probe is shown in Figure 6 This is the distance indicated by segment b. After digestion with lambda exonuclease, the "control" amplicon is incubated with its corresponding signal probe. The amplicon generated by using the signal primer is digested with lambda exonuclease and is not incubated with the signal probe. The amplicon is then loaded onto the detection zone of the GenMark Respiratory Panel cassette (Carlsbad, CA). For various targets, the signal generated from the amplicon using the signal primer is generally equal to or higher than the signal generated using the control primer ( Fig.12 ).

Claims

1. A method for detecting the presence or absence of a target nucleic acid in a sample, the method comprising: a. combining a solution containing a target nucleic acid or suspected of containing a target nucleic acid with an amplification reagent to amplify the target nucleic acid in the presence of the target nucleic acid, the amplification reagent comprising a labeled primer capable of hybridizing with the target nucleic acid; b. amplifying the target nucleic acid in the presence of the target nucleic acid to produce a double-stranded labeled amplicon; c. incubating the double-stranded labeled amplicon with an exonuclease to form a single-stranded labeled amplicon; d. hybridizing the single-stranded labeled amplicon to a first capture probe; and e. Detecting the presence or absence of the target nucleic acid in the sample using electrochemical detection.

2. The method of claim 1, wherein the labeled primer, the double-stranded labeled amplicon and / or the single-stranded labeled amplicon comprises at least one label selected from the group consisting of ferrocene, methylene blue or osmium.

3. The method according to any one of claims 1 to 2, wherein the labeled primer comprises a ferrocene label on the 5′-end, on any one of nucleotides 1 to 3 at the 5′-end, on the 3′-end, on any one of nucleotides 1 to 3 at the 3′-end, or on both the 5′-end and the 3′-end.

4. The method according to any one of claims 1 to 2, wherein the labeled primer comprises a ferrocene label inside the labeled primer.

5. The method according to any one of claims 1 to 4, wherein the labeled primer, the double-stranded labeled amplicon and / or the single-stranded labeled amplicon comprises a plurality of ferrocene labels at at least two positions.

6. The method according to any one of claims 1 to 5, wherein the single-stranded labeled amplicon comprises a first segment capable of hybridizing with the first capture probe, a second segment incapable of binding to the capture probe, and a third segment comprising at least one label.

7. The method of claim 6, wherein the label is selected from the group consisting of ferrocene, methylene blue or osmium.

8. The method according to any one of claims 1 to 7, wherein the labeled primer comprises a first segment capable of hybridizing to the first capture probe, a second segment comprising a linker, and a third segment comprising at least one label, wherein the linker connects the first segment and the third segment.

9. The method according to any one of claims 1 to 8, further comprising: The labeled primer and the single-stranded labeled amplicon are separated prior to electrochemical detection.

10. The method according to any one of claims 1 to 8, further comprising: The labeled primer and the double-stranded labeled amplicon are separated prior to electrochemical detection.

11. The method according to any one of claims 1 to 10, wherein the labeled primer consists of a first segment capable of hybridizing to the capture probe and a second segment comprising at least a first label.

12. A method for detecting the presence or absence of a target nucleic acid in a sample, the method comprising: a. Receive samples; b. extracting nucleic acid from the sample, wherein the nucleic acid is suspected of containing a target nucleic acid; c. combining the nucleic acid with an amplification reagent to amplify the target nucleic acid in the presence of the target nucleic acid, the amplification reagent comprising a signal primer capable of hybridizing to the target nucleic acid; d. amplifying the target nucleic acid in the presence of the target nucleic acid to produce a double-stranded signal amplicon; e. incubating the double-stranded signal amplifier with an exonuclease to form a single-stranded signal amplifier; f. hybridizing the single-stranded signal amplifier with a first capture probe; and g. Using electrochemical detection to detect the presence or absence of the target nucleic acid in the sample.

13. The method of claim 12, wherein the signal primer comprises an electrochemically detectable label.

14. The method according to any one of claims 12 to 13, wherein the signal primer comprises at least one label selected from the group consisting of ferrocene, methylene blue or osmium.

15. The method of any one of claims 12 to 14, wherein the signal primer does not bind to the first capture probe.

16. The method of any one of claims 12 to 15, wherein a first portion of the single-stranded signal amplifier binds to the first capture probe, and a second portion of the single-stranded signal amplifier binds to a second capture probe.

17. A method for detecting the presence of a target single-stranded or double-stranded nucleic acid in a sample, the method comprising the steps of: a. Provide i. a sample suspected of containing the target nucleic acid; ii. a nucleic acid primer comprising:

1. a nucleic acid sequence complementary to at least a portion of the target nucleic acid, and 2. a first electrochemically detectable label; iii. Reagents for nucleic acid chain extension; b. forming a reaction mixture comprising (i), (ii) and (iii) above; c. contacting the nucleic acid primer with the target nucleic acid in the presence of the target nucleic acid under hybridization conditions; d. extending the nucleic acid primer in the presence of the target nucleic acid, thereby incorporating the electrochemically detectable label into the amplicon to form an electrochemically labeled amplicon; e. denaturing the electrochemically labeled amplicon; f. hybridizing the electrochemically labeled amplicon with a capture probe bound to an electrode surface; g. Detecting the presence of the target nucleic acid by detecting energy transfer between the electrochemically labeled amplicon and the electrode surface.

18. The method of claim 17, wherein the electrochemically detectable label is selected from the group consisting of ferrocene, methylene blue or osmium.

19. The method of any one of claims 17 to 18, wherein the nucleic acid primer comprises a first portion that binds to the capture probe and a second portion that does not bind to the capture probe.

20. The method of any one of claims 17 to 19, wherein the nucleic acid primer further comprises a second electrochemically detectable label, and wherein the first electrochemically detectable label and the second electrochemically detectable label are different.

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