Detection of nucleic acids by strand invasion based amplification
Inventors
Eboigbodin, Kevin • Brummer, Mirko
Assignees
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Publication Number
US-11008625-B2
Publication Date
2021-05-18
Expiration Date
Abstract
A method for detecting a target nucleic acid sequence in a sample in the presence of at least protein capable of binding to single-stranded DNA is provided, comprising contacting said sample with at least one oligonucleotide probe comprising a fluorophore, a quencher and a region complementary to said target nucleic acid sequence. The sequence of the oligonucleotide probe comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides.
Core Innovation
The invention provides a method for detection of a target nucleic acid sequence in a sample in the presence of at least one recombinase. The method contacts the sample with at least one oligonucleotide probe comprising a fluorophore, a quencher, and a region complementary to the target nucleic acid sequence, wherein the probe sequence comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides, and the probe is single stranded.
The method further performs strand invasion based amplification of the target nucleic acid sequence under isothermal conditions. This amplification includes contacting the sample with at least one upstream primer and at least one downstream primer, each comprising a region complementary to the target nucleic acid sequence, and a strand invasion oligonucleotide comprising a region complementary to the target nucleic acid sequence.
The strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single stranded to allow binding of the upstream primer, the downstream primer, and the probe. Recombinase mediated strand invasion of the double stranded target nucleic acid sequence is used to promote amplification, and the probe design includes one or more modified RNA nucleotides selected from 2′-fluoro ribonucleotides, 2′-O-methyl ribonucleotides and/or LNA ribonucleotides.
The method is directed to maintaining probe quenching until the appropriate complementary template or amplicon is present, thereby addressing false or irrelevant activation in the presence of single-stranded DNA binding proteins.
Claims Coverage
The document provides one independent claim covering probe-based strand invasion detection combined with isothermal strand invasion based amplification, with dependent claims refining probe composition and detection constraints. The core inventive coverage is centered on a fluorophore/quencher single-stranded probe with substantial RNA/modified RNA/PNA content, and its use in a recombinase-driven strand invasion and isothermal amplification workflow.
Fluorophore-quencher single-stranded probe with substantial RNA-modified content
A method in which the sample is contacted with at least one oligonucleotide probe comprising a fluorophore, a quencher, and a region complementary to the target nucleic acid sequence, wherein the probe sequence comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides and the probe is single stranded.
Recombinase-driven strand invasion and isothermal amplification workflow
A method that performs strand invasion based amplification of the target nucleic acid sequence under isothermal conditions, comprising contacting the sample with at least one upstream primer and at least one downstream primer, each having a region complementary to the target nucleic acid sequence, and a strand invasion oligonucleotide with a region complementary to the target nucleic acid sequence, wherein the strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single stranded to allow binding of the upstream primer, the downstream primer, and the probe, and wherein the recombinase is used for strand invasion of the target nucleic acid sequence.
Defined modified RNA nucleotide selection in the probe
A method in which the oligonucleotide probe comprises one or more modified RNA nucleotides selected from 2′-fluoro ribonucleotides, 2′-O-methyl ribonucleotides and/or LNA ribonucleotides.
Overall claim coverage is centered on detecting a target nucleic acid sequence using a single-stranded fluorophore/quencher oligonucleotide probe with at least 20% RNA nucleotides, including specified modified RNA nucleotides and/or PNA, in combination with recombinase-mediated strand invasion and isothermal strand invasion based amplification using upstream and downstream primers plus a strand invasion oligonucleotide that renders the double-stranded target single stranded for primer and probe binding.
Stated Advantages
Addresses false or irrelevant probe activation in the presence of single-stranded DNA binding proteins.
Maintains probe quenching unless a complementary template or amplicon is present.
Documented Applications
Diagnostic detection of pathogen-derived nucleic acid targets, including toxigenic C. difficile and S. typhimurium.
Multiplex detection using the probe and amplification concepts described.
Use in kit/composition formulations for nucleic-acid detection based on the described probe and isothermal strand invasion amplification approach.
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