Nucleic acid detection method
Inventors
Lamble, Henry John • Lloyd, David • Dunski, Eryk • Watson, Steven
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12275991-B2
Publication Date
2025-04-15
Expiration Date
Abstract
The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.
Core Innovation
The invention relates to a method for detecting the presence of a single stranded target nucleic acid of defined sequence in a sample without temperature cycling. The method produces, in the presence of the target nucleic acid, an amplification product using strand displacement DNA polymerase, dNTPs, and one or more modified dNTP. The amplification is coupled to the subsequent production of a detector species for signal generation, rather than directly reading the amplification product itself.
The amplification product is produced by contacting the sample with a first oligonucleotide primer and a second oligonucleotide primer that include strand-specific restriction enzyme recognition sequence and cleavage site regions, together with first and second restriction enzymes that are not nicking enzymes. The first restriction enzyme cleaves only the first primer strand at a double stranded recognition sequence and cleavage site, while cleavage of the reverse complementary strand is blocked due to modifications incorporated into the reverse complementary strand by the DNA polymerase using the modified dNTP.
Instead of detecting the amplification product directly, the amplification product is contacted with a first oligonucleotide probe and a second oligonucleotide probe. The probes hybridise to first and second single stranded detection sequences in at least one species within the amplification product to produce a detector species, wherein one or both probes includes a repeat motif sequence comprising 3 or more repeat copies of a 2 to 4 base DNA sequence motif. One of the first and second probes can be blocked at the 3′ end from extension by the DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, with the blocked oligonucleotide probe contacted simultaneously to the performance of the amplification step.
Detection of the detector species is achieved by contacting the detector species with a substrate having an immobilised oligonucleotide capture probe comprising a single stranded hybridisation sequence complementary to the repeat motif sequence. The presence of the detector species indicates the presence of the target nucleic acid in the sample, enabling intrinsic coupling of isothermal amplification to downstream detection.
Claims Coverage
One independent claim is identified (clm-00001). It includes inventive features spanning isothermal amplification without temperature cycling, strand displacement DNA polymerase combined with restriction enzymes that are not nicking enzymes, blocking reverse-complement cleavage via modified dNTPs, and probe-based production of a detector species coupled to detection using an immobilised capture probe and a repeat motif sequence.
Isothermal amplification without temperature cycling using strand displacement, restriction enzymes not nicking enzymes, and modified dNTPs that block reverse-complement cleavage
A method for detecting a single stranded target nucleic acid of defined sequence by producing, without temperature cycling, an amplification product using strand displacement DNA polymerase, dNTPs, one or more modified dNTP, first and second oligonucleotide primers containing restriction enzyme recognition sequence and cleavage site regions, and first and second restriction enzymes that are not nicking enzymes and cleave only the corresponding primer strand when the recognition sequence and cleavage site are double stranded, wherein cleavage of the reverse complementary strand is blocked due to modifications incorporated into the reverse complementary strand by the DNA polymerase using the one or more modified dNTP.
Coupled detector species generation using first and second oligonucleotide probes that hybridise to detection sequences in amplification product including a repeat motif
A method in which the amplification product is contacted with a first oligonucleotide probe hybridising to a first single stranded detection sequence in at least one species within the amplification product and attached to a moiety that permits its detection, and a second oligonucleotide probe hybridising to a second single stranded detection sequence upstream or downstream of the first detection sequence in said at least one species and comprising a repeat motif sequence comprising 3 or more repeat copies of a 2 to 4 base DNA sequence motif, wherein hybridisation of the first and second probes to said at least one species produces a detector species.
Probe blocking and non-cleavability of a 3′-blocked probe to support detector species formation
A method wherein one of the first and second oligonucleotide probes is blocked at the 3′ end from extension by the DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes, and wherein the blocked oligonucleotide probe is contacted with the sample simultaneously to the performance of step a).
Detection via substrate with immobilised oligonucleotide capture probe complementary to repeat motif
A method in which detecting the detector species comprises contacting the detector species with a substrate having an immobilised oligonucleotide capture probe comprising a single stranded hybridisation sequence complementary to the repeat motif sequence, wherein the presence of the detector species indicates the presence of the target nucleic acid in the sample.
The independent claim covers an isothermal, temperature-cycling-free detection method that couples strand displacement amplification using restriction enzymes (not nicking enzymes) with modified dNTP-mediated blocking of reverse-complement cleavage to the generation of a detector species using two oligonucleotide probes including a repeat motif, followed by detection using an immobilised capture probe complementary to the repeat motif.
Stated Advantages
Improved sensitivity/specificity versus prior NEAR/nicking-enzyme approaches [stated in partial content].
Rapid low-cost point-of-care formats, especially nucleic acid lateral flow [stated in partial content].
Intrinsic coupling of amplification to detection [stated in partial content].
Faster/stronger signals than WO2014/164479 [stated in partial content].
Documented Applications
Nucleic acid lateral flow detection using an immobilized oligonucleotide capture probe, including performance using a nucleic acid lateral flow strip [stated in partial content].
Detection in clinical specimens, including RNA virus detection exemplified with nasopharyngeal swab [stated in partial content].
Detection of RNA viruses and specific pathogens including RNA viruses listed as HIV, influenza, RSV, Rhinovirus, norovirus, HPV, Ebola, or Epstein-Barr virus [stated in partial content].
Interested in licensing this patent?