The present invention provides a complex of LNA probe and graphene oxide, and a nucleic acid detection method using the same. In the present invention, LNA-containing molecular beacon is conjugated through covalent bonding with graphene oxide, a single strand of the molecular beacon binds to a target nucleic acid to form a complex, and the complex is separated from graphene oxide to induce a fluorescence signal. The molecular beacon and graphene oxide can be covalently bonded to minimize non-specific signals, and a LNA-added molecular beacon is designed in a double strand to detect a very low concentration of target nucleic acid with high sensitivity, as well as a fluorescent signal, and the multiple target nucleic acids can be detected simultaneously through diversification of the fluorescent signal to enable easy and accurate detection of a nucleic acid biomarker whose specific expression level is specifically changed according to diseases and disease progression.

Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.

Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.

Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.

A method and kit for the capture and purification of specific nucleic acids from a sample with affinity capture probes on a solid support and for the replication of said nucleic acids with a strand displacing polymerase, whereby a second primer complementary to a sequence in each of the target nucleic acids distinct from that bound by capture probes is also bound to the nucleic acid targets, and extension of one of the primers on each target effects the separation of the copied nucleic acid strands from the solid support. Incorporation of universal nucleic acid sequences during their replication enables the simultaneous and highly specific amplification of multiple nucleic acid target sequences with minimal production of artifacts.

A method and kit for the capture and purification of specific nucleic acids from a sample with affinity capture probes on a solid support and for the replication of said nucleic acids with a strand displacing polymerase, whereby a second primer complementary to a sequence in each of the target nucleic acids distinct from that bound by capture probes is also bound to the nucleic acid targets, and extension of one of the primers on each target effects the separation of the copied nucleic acid strands from the solid support. Incorporation of universal nucleic acid sequences during their replication enables the simultaneous and highly specific amplification of multiple nucleic acid target sequences with minimal production of artifacts.

Disclosed herein are methods and compositions for detecting amplicon generated using loop-mediated amplification (LAMP). In particular, the invention relates to compositions comprising molecular beacons and/or LAMP primers and methods for generating and detecting LAMP amplicons. In particular, molecular beacons that bind to novel regions of a LAMP amplicon, and methods of using these probes, are provided.

Disclosed herein are methods and compositions for detecting amplicon generated using loop-mediated amplification (LAMP). In particular, the invention relates to compositions comprising molecular beacons and/or LAMP primers and methods for generating and detecting LAMP amplicons. In particular, molecular beacons that bind to novel regions of a LAMP amplicon, and methods of using these probes, are provided.

Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.

Disclosed herein are primers and probes related to the detection of SARS-CoV-2 via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of SARS-CoV-2 in test samples and/or to diagnose Covid-19. Specifically, the present disclosure describes primers and probes that bind to the N gene, ORF1ab, or E gene of SARS-CoV-2 coronavirus for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.