Nine-Type HPV Nucleic Acid Detection With Thermal Multiplexing
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Solution Overview
Problem
Conventional real-time detection methods are limited in their ability to simultaneously detect multiple HPV types due to the restriction of available fluorescent labels and the complexity of melting analysis, making it difficult to efficiently genotype HPV in a single reaction.
Innovation Solution
A method involving the use of at least nine oligonucleotide sets, each comprising an amplifying and signaling oligonucleotide with distinct fluorescent labels, allowing for the detection of at least nine HPV types by measuring signals at three different temperatures using three detection channels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional real-time detection methods using labeled probes or primers are used, then detection of target nucleic acid can be achieved, but the number of target nucleic acids that can be simultaneously detected is limited to the number of available fluorescent labels
Solution Approach 1:
A single fluorescent label system is designed to perform multiple detection functions by utilizing different detection temperatures. The same fluorescent label can detect multiple HPV types by measuring signals at different temperatures, making the detection system universal rather than requiring separate labels for each target
Solution Approach 2:
The detection approach transitions from using multiple fluorescent labels (one dimension: spectral multiplexing) to using a single fluorescent label with multiple detection temperatures (another dimension: thermal multiplexing). This dimensional shift allows detection of multiple HPV types without increasing the number of fluorescent labels
2Quantity of substance
If melting analysis is used to detect multiple target nucleic acids, then multiple HPV types can be detected, but the detection process takes longer and design of probes with different Tm values becomes increasingly difficult
Solution Approach 1:
The invention merges target amplification and target detection into a single real-time process. The signaling oligonucleotide generates a signal during the amplification reaction itself, eliminating the need for a separate melting analysis step and reducing total detection time
Solution Approach 2:
The invention replaces the mechanical/thermal melting analysis process with a chemical signaling mechanism. Instead of relying on temperature-dependent probe dissociation (melting), the system uses enzymatic cleavage of the signaling oligonucleotide to generate fluorescent signals, which can be detected in real-time during amplification
3Productivity
If conventional real-time detection methods are used, then detection can be performed in real-time, but simultaneous detection of multiple HPV types in one reaction is limited
Solution Approach 1:
The invention changes the detection parameter from fluorescent label wavelength to detection temperature. By monitoring signal intensity at different temperatures with a single fluorescent label, the system achieves multiplexing capability while maintaining real-time detection efficiency
Solution Approach 2:
The detection process is segmented into temperature-specific signal measurements. Each HPV type is assigned to specific detection temperature(s), allowing simultaneous detection of multiple types through temporal segmentation of the detection process rather than requiring spatial or spectral separation
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
Enables the simultaneous detection of at least nine HPV types in a single reaction vessel without the need for melting analysis, providing a cost-effective and time-saving solution for HPV genotyping.
Implementation Method 1
a signaling oligonucleotide having a fluorescent label linked thereto, which serves to generate a signal in the presence of a target nucleic acid
Implementation Method 2
an amplifying oligonucleotide, which serves to amplify a target nucleic acid of an HPV type of interest
Data Source
AI summary
The present method allows for detection of at least nine HPV types in a real-time manner in one reaction vessel by analyzing signals measured at three temperatures using at least three different types of fluorescent labels. In particular, the method of the present invention can detect at least nine target nucleic acids in a real-time manner without performing a melting analysis.