Isothermal based-dual functional oligonucleotide including reporter dye, and quencher for isothermal nucleic acid amplification and measurement methods using
Inventors
WON, Yoo Deok • Park, Hae Joon • SEONG, Hyo Jin • LIM, Mi Ae • Lee, Sun Young
Assignees
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Publication Number
US-11795514-B2
Publication Date
2023-10-24
Expiration Date
Abstract
The invention relates to an isothermal-based dual functional oligonucleotide containing quencher and reporter dye for an isothermal nucleic acid amplification and a method for nucleic acid amplification and measurement using the same. The present invention is directed to a method capable of obviating the need for an additional oligonucleotide, in addition to four to six types of oligonucleotides for a nucleic acid amplification reaction of LAMP, detecting the amount of fluorescence according to the amplification of the nucleic acid of target gene—specific sequence for DNA and RNA, enabling the detection also after the completion of the reaction, and detecting the amount of fluorescence in real-time. Therefore, the present invention allows simultaneous multiple tests by measuring the amount of fluorescence in one tube after the completion of the reaction or in real time while varying the reporter dye according to the target gene.
Core Innovation
The document describes an isothermal nucleic acid amplification detection system using loop-mediated isothermal nucleic acid amplification (LAMP) and reverse transcription LAMP (RT-LAMP). It uses a dual functional LAMP oligonucleotide in which primer and probe functions are integrated within a single oligonucleotide for a specific sequence of a target gene, enabling real-time fluorescence monitoring and end-point fluorescence detection without opening reaction tubes.
The dual functional oligonucleotide is constructed by incorporating internal bases, such as internal dT, internal dA, internal dC, internal dG, internal dU, or internal dR, and by positioning a reporter dye and a quencher within a specified mer interval plus one or more bubble structures. The reported design parameters include a reporter-quencher mer distance in the range of 21-33 mer and a melting temperature range of 30-70 °C.
The system includes forward inner primer (FIP) and reverse inner primer (RIP) oligonucleotides formed with these dual functional elements. The document further describes that the dual functional LAMP approach supports DNA and RNA targets and can be implemented as one-step RT-LAMP, with multiplexing by varying reporter dyes for different targets.
Claims Coverage
The provided portion contains one independent claim set centered on dual-functional LAMP/RIP/FIP oligonucleotides for use in LAMP to amplify a specific gene sequence, limited to enumerated sequences. A dependent claim refines the scope by defining a kit for specimen gene amplification and specifying which enumerated oligonucleotides are included.
Enumerated dual-functional LAMP oligonucleotides for targeted LAMP amplification
The oligonucleotide is one of oligonucleotides set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 15, or SEQ ID NO: 16 for use in a loop-mediated isothermal nucleic acid amplification (LAMP) reaction for a specific sequence of a target gene.
Specimen gene amplification kit comprising the specified enumerated oligonucleotides
A kit for amplifying a gene in a specimen that includes the oligonucleotides according to the claim, specifically comprising oligonucleotides set forth in either SEQ ID NOs: 5-8 or SEQ ID NOs: 15-16.
Overall, the claim set is centered on using enumerated dual-functional LAMP oligonucleotide sequences, including FIP and RIP variants, for targeted LAMP amplification, and on packaging either the SEQ ID NOs 5-8 set or the SEQ ID NOs 15-16 set into a specimen gene amplification kit.
Stated Advantages
Enables real-time fluorescence monitoring and end-point fluorescence detection.
Supports detection without opening reaction tubes.
Enables DNA/RNA detection using LAMP and reverse transcription LAMP (RT-LAMP), including one-step RT-LAMP.
Allows multiplexing by varying reporter dyes for different targets.
Documented Applications
Detection and amplification examples are described using Ebola virus (Bundibugyo and Reston segments), with fluorescence behavior dependence on reporter-quencher distance and on forward/reverse constructs.
Multiplexing is described using different reporter dyes for different targets.
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