Dual oligonucleotide method of nucleic acid detection

A method for detecting the presence or absence of a target nucleic acid in a biological sample, said method comprising:

• (a) contacting said biological sample with a set of oligonucleotides to produce a fluorescently labeled amplicon if said target nucleic acid is present in said sample, said set of oligonucleotides comprising:
• i) a first oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said first oligonucleotide, wherein said 3′ sequence is complementary to a first portion of said target nucleic acid, and wherein a fluorescent moiety is attached in the 5′ nucleotide sequence of said first oligonucleotide;
• and
• ii) a second oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said second oligonucleotide, wherein said 3′ nucleotide sequence is complementary to a second portion of said target nucleic acid, and wherein an acceptor moiety is attached in the 5′ nucleotide sequence of said second oligonucleotide;
• wherein said 5′ nucleotide sequence of said first oligonucleotide is at least 80% identical to the complement of said 5′ nucleotide sequence of said second oligonucleotide, and wherein the 3′ nucleotide sequence of each said oligonucleotide is not labeled with a fluorescent moiety or an acceptor moiety that is compatible with said fluorescent moiety attached to said first oligonucleotide or said acceptor moiety attached to said second oligonucleotide;
• and
• (b) detecting fluorescence of said amplicon.

A kit for detecting a nucleic acid comprising a set of oligonucleotides, said set of oligonucleotides comprising:

• a) a first oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said first oligonucleotide, wherein said 3′ sequence is at least 80% identical to a first portion of said target nucleic acid, and wherein a fluorescent moiety is attached in the 5′ nucleotide sequence of said first oligonucleotide;
• and
• b) a second oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said second oligonucleotide, wherein said 3′ nucleotide sequence is at least 80% identical to a second portion of said target nucleic acid, and wherein an acceptor moiety is attached in the 5′ nucleotide sequence of said second oligonucleotide;
• wherein said 5′ nucleotide sequence of said first oligonucleotide is at least 80% identical to the complement of said 5′ nucleotide sequence of said second oligonucleotide, and wherein the 3′ nucleotide sequence of each said oligonucleotide is not labeled with a fluorescent moiety or an acceptor moiety that is compatible with said fluorescent moiety attached to said first oligonucleotide or said acceptor moiety attached to said second oligonucleotide;
• wherein said first oligonucleotide or second oligonucleotide comprises a modified base.

A reaction mixture for fluorescence-based monitoring of a polymerase chain reaction, said reaction mixture comprising a set of oligonucleotides, a DNA polymerase, dNTPs, and MgCl2, wherein said set of oligonucleotides comprises:

• a) a first oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said first oligonucleotide, wherein said 3′ sequence is at least 80% identical to a first portion of said target nucleic acid, and wherein a fluorescent moiety is attached in the 5′ nucleotide sequence of said first oligonucleotide;
• and
• b) a second oligonucleotide comprising a 5′ and 3′ nucleotide sequence, wherein said 5′ nucleotide sequence is 5-23 nucleotides in length beginning at the 5′ end of said second oligonucleotide, wherein said 3′ nucleotide sequence is at least 80% identical to a second portion of said target nucleic acid, and wherein an acceptor moiety is attached in the 5′ nucleotide sequence of said second oligonucleotide;
• wherein said 5′ nucleotide sequence of said first oligonucleotide is at least 80% identical to the complement of said 5′ nucleotide sequence of said second oligonucleotide, and wherein the 3′ nucleotide sequence of each said oligonucleotide is not labeled with a fluorescent moiety or an acceptor moiety that is compatible with said fluorescent moiety attached to said first oligonucleotide or said acceptor moiety attached to said second oligonucleotide.