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US 2026/0258477 A1Published application
Dual quenching assay for multiplex detection of target nucleic acids
AssigneeGrifols Diagnostic Solutions Inc.
InventorsUffe Vest SCHNEIDER, Søren Morgenthaler Echwald, Nikolaj Dam Mikkelsen
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Abstract
The present invention relates to a method for detecting at least one target nucleic acid sequence from a nucleic acid mixture by a double quenched assay. The double quenched assay of the method exploits a novel approach for melting temperature mediated identification of multiple target nucleic acid sequences. The invention further relates to a kit of parts.
Claims19
A method for detecting a nucleic acid sequence, comprising:
- (a) hybridizing a nucleic acid sequence with a probe comprising (i) a targeting portion comprising a nucleotide sequence substantially complementary to the nucleic acid sequence, and (ii) a melting temperature deciding region (MTDR) comprising a nucleotide sequence non-complementary to the nucleic acid sequence, and (iii) at least one set of interactive labels comprising at least one fluorophore and at least one quencher;
- (b) hybridizing said probe with a quenching probe comprising (i) a capturing portion comprising a nucleotide sequence which is reverse complementary to the MTDR of the probe and (ii) at least one quenching molecule, wherein the MTDR of the probe is configured to hybridize with the capturing portion of the quenching probe to form a tag duplex;
- (c) contacting the tag duplex with an enzyme having nuclease activity to induce cleavage of the tag duplex and release of an activated tag duplex fragment comprising the MTDR hybridized to the capturing portion and the at least one fluorophore;
- (d) melting or hybridizing said activated tag duplex fragment to obtain a signal from the at least one fluorophore, and
- (e) detecting the signal to determine presence of the nucleic acid sequence.
2 more elements
A kit of parts for detecting at least one nucleic acid sequence from a nucleic acid mixture, the kit comprising:
- i. at least one probe comprising (i) a targeting portion comprising a nucleotide sequence substantially complementary to a nucleic acid sequence in the nucleic acid mixture, and (ii) a melting temperature deciding region (MTDR) comprising a nucleotide sequence non-complementary to the nucleic acid sequence, and (iii) at least one set of interactive labels comprising a fluorophore and a quencher, and
- ii. at least one quenching probe comprising (i) a capturing portion comprising a nucleotide sequence which is reverse complementary to the MTDR and (ii) a quencher, and
- iii. optionally instructions for detecting the nucleic acid sequence.
A reaction mixture, comprising:
- at least one pair of primers, at least one probe and at least one quenching probe,
- wherein said pair of primers comprises a first a primer complementary to a nucleic acid and which primes synthesis of a first extension product that is complementary to the nucleic acid, and a second primer complementary to the first extension product and which primes synthesis of a second extension product;
- wherein said at least one probe comprises (i) a targeting portion comprising a nucleotide sequence substantially complementary to the nucleic acid or to the complement of the nucleic acid, and (ii) a melting temperature deciding region (MTDR) comprising a nucleotide sequence non-complementary to the nucleic acid, and (iii) at least one set of interactive labels comprising a fluorophore and a quencher;
- and
- wherein said at least one quenching probe comprises (i) a capturing portion comprising a nucleotide sequence which is reverse complementary to the MTDR and (ii) a quencher;
- wherein the targeting portion hybridizes to the nucleic acid in a region between one member of said primer pair and the complement of the other member of said primer pair.
3 more elements
A method for detecting a nucleic acid sequence, comprising:
- (a) hybridizing a nucleic acid sequence with a probe comprising (i) a targeting portion comprising a nucleotide sequence substantially complementary to the nucleic acid sequence, and (ii) a melting temperature deciding region (MTDR) comprising a nucleotide sequence non-complementary to the nucleic acid sequence, and (iii) at least one set of interactive labels comprising a fluorophore and a quencher;
- (b) contacting the hybridized nucleic acid sequence-probe with an enzyme having nuclease activity to induce cleavage of a fragment comprising the MTDR and the at least one fluorophore;
- (c) hybridizing the fragment with a quenching probe to form an activated tag duplex, the quenching probe comprising (i) a capturing portion comprising a nucleotide sequence which is reverse complementary to the MTDR and (ii) a quencher;
- (d) melting or hybridizing the activated tag duplex to obtain a signal from the fluorophore, and
- (e) detecting the signal to determine presence of the nucleic acid sequence.
2 more elements
Dependencies are inferred from the claim text.