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US 11,685,960 B2Granted
Rapid nucleic acid detection without sample preparation
AssigneeCrosslife Technologies Inc.
InventorsHyunDae Cho, Yuanyuan Wu, Jarred Yasuhara-Bell
Life of this patent
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Abstract
Sensitive and specific detection of nucleic acids can be achieved using a chemical ligation-based template assisted rapid assay (TARA-L) with simple chemical reactions between probes and without the need for enzymes. Probes are designed to form a ligation product when they anneal to adjacent portions of a target nucleic acid. The ligation products can be detected, such as in immunochromatographic assays. The methods allow for the fast, efficient analysis of biological samples for the presence of nucleic acids and can be used, for example, in point of care settings.
Claims28
A method of determining the presence of a target nucleic acid in a sample, the method comprising:
- contacting the sample with a reaction mixture comprising at least one set of chemically-reactive probes, the at least one set of chemically-reactive probes comprising:
- a plurality of a first probe, the first probe comprising a first nucleic acid region that is complementary to a first part of the target nucleic acid sequence and a first chemical group at a 3′end of the first nucleic acid region selected from a 2-Fluoro-5-nitropyridine (FNP) group, a 1-Fluoro-2,4,6-Trinitrobenzene (FNTB) group, a succinimidyl iodoacetate (SIA) group, an amine (NH2) group, thiophosphate (PS) group, or hydroxyl (OH) group, and
- a plurality of a second probe, the second probe comprising a second nucleic acid region complementary to a second, different part of the target nucleic acid sequence and a second, different chemical group at a 5′end of the second nucleic acid region selected from a 2-Fluoro-5-nitropyridine (FNP) group, a 1-Fluoro-2,4,6-Trinitrobenzene (FNTB) group, a succinimidyl iodoacetate (SIA) group, an amine (NH2) group, a thiophosphate (PS) group, or a hydroxyl (OH) group,
- wherein the first and second parts of the target nucleic acid sequence are separated by 10 nucleotides or less,
- wherein in the presence of the target nucleic acid in the sample the first probe and second probe anneal to the first and second parts of the target nucleic acid sequence and ligate together through a chemical ligation reaction between the first chemical group and the second chemical group to form a chemically ligated product;
- and
- detecting the chemically ligated product by capturing and visualizing the chemically ligated product on an immunochromatographic test strip,
- wherein detection of the chemically ligated product indicates the presence of the target nucleic acid in the sample, and
- wherein:
- when the first chemical group at a 3′end of the first nucleic acid sequence is 2-Fluoro-5-nitropyridine (FNP) or a 1-Fluoro-2,4,6-Trinitrobenzene (FNTB) group, the second, different chemical group at a 5′end of the second nucleic acid, is an amine (NH2), thiophosphate (PS), or hydroxyl (OH) group,
- when the first chemical group at a 3′end of the first nucleic acid sequence is an amine (NH2), thiophosphate (PS), or hydroxyl (OH) group, the second, different chemical group at a 5′end of the second nucleic acid, is a 2-Fluoro-5-nitropyridine (FNP) group or a 1-Fluoro-2,4,6-Trinitrobenzene (FNTB) group,
- when the first chemical group at a 3′end of the first nucleic acid sequence is a hydroxyl (OH) group, the second, different chemical group at a 5′end of the second nucleic acid, is a succinimidyl iodoacetate (SIA) group, and
- when the first chemical group at a 3′end of the first nucleic acid sequence is a succinimidyl iodoacetate (SIA) group, the second, different chemical group at a 5′end of the second nucleic acid, is a hydroxyl (OH) group.
10 more elements
A method of diagnosing a condition in a subject, the method comprising:
- obtaining a sample from the subject that may comprise a target nucleic acid associated with the condition;
- contacting the sample with a reaction mixture comprising at least one set of probes, the at least one set of probes comprising:
- a plurality of a first probe comprising a first nucleic acid region that is complementary to a first part of the target nucleic acid sequence and a first thiophosphate group at a 3′ end, and
- a plurality of a second probe, the second probe comprising a second nucleic acid region complementary to a second, different part of the target nucleic acid sequence and a 2-Fluoro-5-nitropyridine (FNP) group or a 1-Fluoro-2,4,6-Trinitrobenzene (FNTB) group at a 5′ end,
- wherein the first part and the second part of the target nucleic acid sequence are separated by 0 to 10 nucleotides;
- wherein in the presence of the target nucleic acid sequence the first and second products are ligated through a reaction between the thiophosphate group and the primary amine group to form a chemically ligated product;
- and
- detecting the chemically ligated product by immunochromatographic analysis on an immunochromatographic test strip,
- wherein detection of the chemically ligated product diagnoses the condition in the subject.
6 more elements
Dependencies are inferred from the claim text.