Patent ID: 9260746
Filing Date: 2016-02-16
CPC Classification: C12Q

Claim Text:
1. A method of detecting a target nucleic acid molecule, comprising: incubating a sample comprising the target nucleic acid molecule with a forward primer comprising a sequence homologous to the target nucleic acid molecule and a reverse primer comprising a sequence homologous to the target nucleic acid molecule under conditions sufficient to allow amplification of the target nucleic acid molecule; amplifying the target nucleic acid molecule using real-time polymerase chain reaction (PCR), thereby generating a labeled amplicon; denaturing the labeled amplicon; generating a melting curve; and detecting a signal from the label, wherein the forward primer or the reverse primer is linked at its 5′-end to the 3′-end of a photoinduced electron transfer (PET) tag, wherein the PET tag comprises the sequence 5′-AGGCGCATAGCGCCTGG-3′ (SEQ ID NO: 4); 5′-AGGCGCGATACGCGCCTGG-3′ (nucleotides 1 to 19 of SEQ ID NO: 10); 5′-AGGCGCGATCACGCGCCTGG-3′ (nucleotides 1 to 20 of SEQ ID NO: 11); 5′-AGGCGCGATTCACGCGCCTGG-3′ (nucleotides 1 to 21 of SEQ ID NO: 12); 5′-AGGCGATACGCCTGG-3′ (nucleotides 1 to 15 of SEQ ID NO: 23); 5′-AGGCGCATCAGCGCCTGG-3′ (nucleotides 1 to 18 of SEQ ID NO: 24); 5′-AGGCGATCACGCCTGG-3′ (nucleotides 1 to 16 of SEQ ID NO: 25); 5′-AGGCGCATTCAGCGCCTGG-3′ (nucleotides 1 to 19 of SEQ ID NO: 26); or 5′-AGGCGATTCACGCCTGG-3′ (nucleotides 1 to 17 of SEQ ID NO: 27), wherein the 5′-end nucleotide of the PET tag comprises a label, wherein the PET tag comprises a stem and a loop of a stem-loop, wherein the PET tag comprises two consecutive G nucleotides at the 3′-end of the PET tag, wherein the stem-loop brings the label on the 5′-end nucleotide and the two consecutive G nucleotides at the 3′-end of the PET tag into proximity, thereby quenching a detectable signal from the label in the absence of a target nucleic acid sequence, wherein the PET tag does not substantially hybridize to the target nucleic acid sequence recognized by the forward and reverse primers, and wherein the detectable signal from the label is unquenched when the labeled forward or reverse primer is incorporated into the amplicon, wherein an increase in signal detected during the real-time PCR indicates that the target nucleic acid molecule is present in the sample and wherein no significant increase in signal detected during the real-time PCR indicates that the target molecule is not present in the sample, and wherein a single peak detected during generating the melting curve indicates that the target nucleic acid molecule is present in the sample and wherein no single peak detected during generating the melting curve indicates that the target molecule is not present in the sample.