Patent ID: 8889355

Claim:
A method for detecting a target nucleic acid in a sample, comprising: a. contacting the sample with at least one set of at least three oligonucleotide probes for a time and under conditions suitable to form an annealed product, wherein the at least one set of at least three oligonucleotide probes comprises at least one first chimeric oligonucleotide probe, at least one medial oligonucleotide probe, and at least one second chimeric oligonucleotide probe; wherein the at least one first chimeric oligonucleotide probe comprises, in a 5′ to 3′ direction: a primer-specific portion comprising an amplification primer nucleotide sequence; and a target-specific portion, the target-specific portion having: complementarity to a 3′ portion of a preselected sequence of the target nucleic acid, a length of 6 nucleotides to 44 nucleotides, at least one nucleotide analog at one of the six 5′-most nucleotides wherein the nucleotide analog has enhanced affinity for base pairing as compared to a corresponding non-modified nucleotide, and 3′-OH and 2′-OR groups on the 3′-terminal nucleotide, wherein R comprises H or C 1 -C 3 alkyl; wherein the at least one medial oligonucleotide probe comprises a target-specific portion having: a 5′-terminal nucleotide comprising a 5′-phosphate group, complementarity to a portion of the preselected sequence of the target nucleic acid intermediate to the first and the second chimeric oligonucleotide probes, and a 3′-OH group on the 3′-terminal nucleotide; wherein the at least one second chimeric oligonucleotide probe comprises, in a 5′ to 3′ direction: a target-specific portion having: a 5′-terminal nucleotide comprising a 5′-phosphate group, complementarity to a 5′ portion of the preselected sequence of the target nucleic acid, a length of 6 nucleotides to 44 nucleotides, and a primer-specific portion comprising an amplification primer nucleotide sequence; wherein, when the at least one medial oligonucleotide probe and the at least one first and the at least one second chimeric oligonucleotide probes are annealed to the target nucleic acid, the 3′ hydroxyl group of the first chimeric oligonucleotide probe is positioned immediately adjacent to the 5′ phosphate group of the medial probe and the 3′-OH group of the medial probe is positioned immediately adjacent to the 5′ phosphate group of the second chimeric oligonucleotide probe; b. contacting the annealed product with a polypeptide having double-strand dependent ligase activity for a time and under conditions suitable to form a ligated product; and c. detecting the target nucleic acid in the sample by detecting the ligated product, or a surrogate thereof.