Method of Fluorescent Detection of Isothermal Loop-Mediated Amplification (LAMP) of a Target Nucleic Acid, Oligonucleotides and Kits Thereof

A set of oligonucleotides for detecting loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence, the set comprising:

• (a) a first outer primer F3 and a second outer primer B3;
• (b) a first inner primer FIP and a second inner primer BIP, wherein:
• (1) FIP consists of a 3′ nucleic acid sequence F2 and a 5′ nucleic acid sequence F1c and BIP consists of a 3′ nucleic acid sequence B2 and a 5′ nucleic acid sequence B1c;
• (2) F2 is complementary to a F2c region of the target nucleic acid sequence and B2 is complementary to a B2c region of the target nucleic acid sequence;
• and
• (3) F2c and B2c are non-overlapping regions located on opposite strands of the target nucleic acid sequence;
• (c) a loop primer LF and/or a loop primer LB, wherein:
• (1) said loop primer LF is capable of hybridizing to a region of the target nucleic acid sequence between F2 and F1;
• and
• (2) said loop primer LB is complementary to a region of the target nucleic acid sequence between B1 and B2;
• wherein said loop primer LF, or said loop primer LB, if present, is labeled with at least one acceptor fluorophore at its 5′-end;
• and
• (d) a nucleic acid probe, labeled at its 3′-end with at least one donor fluorophore capable of transferring excitation energy to said at least one acceptor fluorophore of a labeled loop primer, wherein:
• (1) if said loop primer LF is labeled with said at least one acceptor fluorophore at its 5′-end, the nucleotide sequence of said nucleic acid probe is selected so that said nucleic acid probe is capable of hybridizing to the target nucleic acid sequence at a position 5′ to the position at which such labeled loop primer LF hybridizes to said target nucleic acid sequence;
• and
• (2) if said loop primer LB is labeled with said at least one acceptor fluorophore at its 5′-end, the nucleotide sequence of said nucleic acid probe is selected so that said nucleic acid probe is capable of hybridizing to the target nucleic acid sequence at a position 5′ to the position at which such labeled loop primer LB hybridizes to said target nucleic acid sequence;
• wherein hybridization of said nucleic acid probe to the target nucleic acid sequence causes the 3′-end of the nucleic acid probe to be in close proximity to the 5′-end of hybridized labeled loop primer, and wherein:
• the intensity of fluorescence emission of the acceptor fluorophore increases upon absorption of the donor fluorophore excitation energy;
• (ii) the ratio of donor and/or acceptor fluorescence intensities of fluorescence changes upon absorption of the donor fluorophore excitation energy;
• or
• (iii) the fluorescence life-time of the acceptor fluorophore changes upon absorption of the donor fluorophore excitation energy.