Method of fluorescent detection of isothermal loop-mediated amplification (LAMP) of a target nucleic acid, oligonucleotides and kits thereof
Inventors
Minnucci, Giulia • Mesturini, Riccardo
Assignees
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Publication Number
US-11827923-B2
Publication Date
2023-11-28
Expiration Date
Abstract
The invention concerns a method for detecting isothermal loop-mediated (LAMP) amplification of a target nucleic acid sequence which is based on the fluorescence resonance energy transfer (FRET) mechanism. The invention also concerns a set of oligonucleotides and a kit adapted for carrying out the LAMP-FRET method of the invention.
Core Innovation
The invention provides a set of oligonucleotides for detecting loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence using fluorescence resonance energy transfer (FRET) readouts based on hybridization-dependent close proximity of labeled components. A nucleic acid probe is labeled at its 3′-end with at least one donor fluorophore, and a loop primer (LF and/or LB) is labeled at its 5′-end with at least one acceptor fluorophore. Upon LAMP intermediate formation, the probe hybridizes such that the probe 3′-end is brought into close proximity to the 5′-end of the labeled loop primer.
The close proximity enables donor-to-acceptor excitation energy transfer, producing an increased acceptor fluorescence emission intensity and/or changes in the ratio of donor and/or acceptor fluorescence intensities and/or a change in acceptor fluorescence lifetime. The loop primer LF is capable of hybridizing to a region of the target nucleic acid sequence between F2 and F1, and the loop primer LB is complementary to a region between B1 and B2. The nucleic acid probe nucleotide sequence is selected so that it hybridizes at a position 5′ to the position where the labeled loop primer hybridizes.
The disclosure evaluates the labeled loop primer and donor/acceptor probe approach as a comparative alternative to prior FRET-based LAMP and to intercalating dye LAMP, reporting earlier threshold times, improved linearity (higher R²), and enhanced specificity attributed to sequence-specific probe/primer labeling. The described system includes outer primers (F3, B3), inner primers (FIP, BIP) and defines primer-region relationships, while also specifying kit-level components including strand-displacing DNA polymerase options and a fluorescence readout configuration compatible with donor/acceptor intensity and fluorescence lifetime measurements.
Claims Coverage
The independent claim covers a labeled oligonucleotide set for LAMP detection of a target nucleic acid sequence using a FRET-like fluorescence readout, with a nucleic acid probe carrying a donor fluorophore at the 3′-end and a labeled loop primer carrying an acceptor fluorophore at the 5′-end. The inventive features are centered on specific primer composition, donor/acceptor labeling positions, and the hybridization-induced close proximity mechanism that changes acceptor fluorescence intensity/ratio and/or acceptor fluorescence lifetime. The dependent claims further narrow fluorophore selections and kit composition by specifying which labeled loop primer is included and by requiring a strand-displacing DNA polymerase with options.
Labeled LAMP primer set with donor/acceptor fluorophores for FRET-like detection
A set of oligonucleotides for detecting loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence comprising outer primers F3 and B3; inner primers FIP and BIP with defined F2/F1c and B2/B1c relationships to complementary target regions on opposite strands; a loop primer LF and/or LB hybridizing between F2 and F1 or complementary between B1 and B2 and labeled with at least one acceptor fluorophore at its 5′-end; and a nucleic acid probe labeled at its 3′-end with at least one donor fluorophore capable of transferring excitation energy to the acceptor fluorophore, where probe hybridization causes the probe 3′-end to be in close proximity to the labeled loop primer 5′-end so that acceptor fluorescence increases and/or the donor/acceptor fluorescence intensity ratio changes and/or the fluorescence life-time changes.
Across the independent claim and its refinements, the claims focus on configuring LAMP primer components so that a 3′-end donor-labeled nucleic acid probe hybridizes at a defined 5′ position relative to a 5′-end acceptor-labeled loop primer, generating a hybridization-dependent acceptor fluorescence increase and/or changes in donor/acceptor fluorescence intensities and/or acceptor fluorescence lifetime.
Stated Advantages
Earlier threshold times compared with prior FRET-based LAMP and intercalating dye LAMP.
Improved linearity (higher R²) relative to comparison approaches.
Enhanced specificity due to sequence-specific probe/primer labeling.
Documented Applications
LAMP detection of specific targets WSSV and MYH11 using the described labeled oligonucleotide designs and fluorescence readouts.
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