US 11,685,960 B2
Rapid nucleic acid detection without sample preparation
HyunDae Cho, Carlsbad, CA (US); Yuanyuan Wu, Carlsbad, CA (US); and Jarred Yasuhara-Bell, Carlsbad, CA (US)
Assigned to CROSSLIFE TECHNOLOGIES INC., Carlsbad, CA (US)
Filed by CROSSLIFE TECHNOLOGIES INC., Carlsbad, CA (US)
Filed on May 6, 2020, as Appl. No. 16/868,403.
Claims priority of provisional application 62/844,917, filed on May 8, 2019.
Prior Publication US 2020/0354800 A1, Nov. 12, 2020
Int. Cl. C12Q 1/6862 (2018.01); C12Q 1/70 (2006.01); G01N 33/543 (2006.01)
CPC C12Q 1/701 (2013.01) [C12Q 1/6862 (2013.01); C12Q 1/702 (2013.01); G01N 33/54366 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/178 (2013.01); G01N 2333/11 (2013.01); G01N 2333/16 (2013.01); G01N 2800/26 (2013.01)] 28 Claims
 
1. 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.