Fluorescence-Based Multiple Melting Analysis for Multiplex Nucleic Acid Detection

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Solution Overview

Problem

Current real-time PCR technologies are limited in their ability to perform multiplex analysis, allowing for the simultaneous analysis of only 4 to 6 target nucleic acids due to limitations in fluorescence channels and primer design complexity.

Innovation Solution

The development of fluorescence-based multiple melting analysis (FMMA) method, which uses target-specific FMMA probe/primer sets labeled with a fluorophore and a quencher, and incorporating a melting temperature control sequence to induce different melting temperatures, enabling the analysis of multiple targets in a single fluorescence channel.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If conventional real-time PCR with fluorescent dyes is used, then fluorescence detection is achieved, but the number of targets that can be analyzed simultaneously is limited to 4-6 due to overlapping wavelengths

Engineering Contradiction:

Improvenumber of target nucleic acids analyzed simultaneouslyVSAvoidnumber of fluorescence channels required

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The invention changes the detection parameter from fluorescence wavelength to melting temperature. By using probes with different melting temperatures but the same fluorescence wavelength, multiple targets can be distinguished in a single channel. The melting temperature is controlled by adjusting the length and GC content of the probe sequences, allowing multiplex detection without requiring multiple fluorescence channels.

Inventive Principle:

Principle #35Parameter changes

2Quantity of substance

If the PTOCE method is used to increase multiplexing capability, then more target nucleic acids can be analyzed, but primer and oligonucleotide design becomes extremely complex with dozens of components

Engineering Contradiction:

Improvenumber of target nucleic acids analyzedVSAvoidprimer design complexity

Core Design Contradiction:

Quantity of substanceVSEase of manufacture

Solution Approach 1:

The invention extracts and utilizes the melting temperature characteristic of probe-target hybrids as the distinguishing feature. Instead of relying on complex primer designs with multiple components, the method uses simple probe sequences with controlled lengths and GC contents to achieve multiplexing. This reduces the design complexity from dozens of primers to a manageable set of probes with differentiated melting temperatures.

Inventive Principle:

Principle #2Taking out (Extraction)

3Quantity of substance

If fluorescent probes with different wavelengths are used for multiplex analysis, then multiple targets can be detected, but the number of available fluorescence channels is limited

Engineering Contradiction:

Improvenumber of targets detected simultaneouslyVSAvoidnumber of fluorescence channels available

Core Design Contradiction:

Quantity of substanceVSAdaptability or versatility

Solution Approach 1:

The invention makes a single fluorescence channel perform multiple detection functions by using probes with different melting temperatures. The same fluorescence wavelength can detect multiple targets simultaneously, as each target's probe produces a distinct melting temperature signal. This allows one channel to serve the function of multiple channels, increasing versatility without requiring additional wavelengths.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method allows for the simultaneous analysis of multiple target nucleic acids, reducing time and cost, and simplifying the PCR process, while also enabling efficient genotyping and identification of multiple alleles or nucleotide sequences.

Implementation Method 1

fluorescence-based multiple melting analysis (FMMA) method, which uses target-specific FMMA probe/primer sets labeled with a fluorophore and a quencher

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

incorporating a melting temperature control sequence to induce different melting temperatures, enabling the analysis of multiple targets in a single fluorescence channel

Methodology Applied

Scientific EffectMelting: Melting

Data Source

PatentUS12209272B2Multiple analysis method for amplicon by using fluorescence-based multiple melting analysis

Publication Date: 2025.01.28 NANOHELIX

AI summary

A method for multiplex analysis of amplification products using fluorescence-based multiple melting analysis is capable of analyzing multiple target nucleic acid sequences simultaneously in real time. A kit for performing the multiplex analysis contains target-specific primer/probe sets. The method and the kit allow a detection of multiple target nucleic acid sequences by a single polymerase chain reaction using a single fluorescence channel, thereby significantly reducing time and cost for multiplex nucleic acid detection. Therefore, the method and kit may be widely used in the companion diagnostic field in which multiple target nucleic acid genes need to be analyzed, the agricultural and livestock field in which multiple alleles need to be analyzed, and the clinical pathology field in which multiple infectious agents need to be analyzed simultaneously.