Multiplex Nucleic Acid Detection via Temperature Kinetics
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Solution Overview
Problem
Current real-time PCR methods are limited in their ability to detect multiple nucleic acid targets in a single tube due to the need for distinct fluorescent labels, restricting the number of targets that can be analyzed simultaneously to no more than four, as existing instruments can only discriminate among a limited number of optical detection channels.
Innovation Solution
A method involving multiple primer/probe sets with unique annealing temperatures and shared reporter labels, allowing for sequential detection and subtraction of signals to differentiate between targets, enabling the detection of multiple nucleic acid targets in a single sample container using a two-step annealing/elongation temperature profile.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple probes with distinct fluorescent labels are used to detect multiple targets, then the number of detectable targets increases, but the device complexity and cost increase due to the need for multiple optical detection channels
Solution Approach 1:
The patent changes the parameter of probe labeling by using the same fluorophore for all probes instead of distinct fluorescent labels. This allows all probes to be detected through a single optical channel, eliminating the need for multiple detection channels while maintaining the ability to distinguish multiple targets through temperature-dependent hybridization kinetics
Solution Approach 2:
The patent makes a single optical detection channel universal by enabling it to detect multiple different probes labeled with the same fluorophore. The detection channel performs the function of distinguishing between multiple targets not through wavelength discrimination but through kinetic analysis of hybridization at different temperatures
2Quantity of substance
If multiple probes with distinct fluorescent labels are used, then multiple targets can be detected simultaneously, but the cost of the assay increases due to requiring multiple expensive fluorescent labels
Solution Approach 1:
The patent changes the labeling parameter from using multiple distinct fluorescent labels to using a single fluorophore for all probes. This dramatically reduces assay cost while maintaining multiplexing capability through temperature-dependent kinetic discrimination rather than spectral discrimination
3Device complexity
If the number of optical detection channels is limited to four, then the instrument is simpler and cheaper, but the number of targets that can be detected in a single reaction is severely limited to three or four
Solution Approach 1:
The patent changes the discrimination parameter from optical wavelength (requiring multiple channels) to hybridization temperature kinetics (detectable through a single channel). By monitoring how probe hybridization varies with temperature, the system can distinguish multiple targets using only one optical detection channel
Solution Approach 2:
The patent adds a temperature dimension to the detection process. Instead of discriminating targets along the wavelength dimension (which requires multiple optical channels), the system uses temperature as an additional dimension to differentiate targets, allowing a single optical channel to detect multiple targets by analyzing kinetic patterns across temperature gradients
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 approach allows for the simultaneous detection of multiple nucleic acid targets in a single tube, overcoming the limitations of current technologies by enabling the analysis of more targets without the need for multiple fluorescent labels, thus aligning with the increasing clinical demands for multiplex analysis.
Implementation Method 1
The probe is preferably labeled with one or more fluorescent moieties, which emit light of a detectable wavelength. Upon hybridizing to the target sequence or its amplicon, the probe exhibits a detectable change in fluorescent emission.
Implementation Method 2
The probe is preferably labeled with one or more fluorescent moieties, which emit light of a detectable wavelength.
Implementation Method 3
The polymerase chain reaction (PCR) has become a ubiquitous tool of biomedical research, disease monitoring and diagnostics. Amplification of nucleic acid sequences by PCR
Implementation Method 4
A typical real-time PCR protocol involves the use of a labeled probe, specific for each target sequence
Data Source
Figure 1A~1D(ii)
Figure 2
AI summary
The disclosure is a single-tube multiplex assay, capable of simultaneously detecting multiple nucleic acid targets, using multiple hybridization primers and probes, labeled with the same fluorescent reporter label, but each having a distinct annealing temperature. The assay can be further multiplexed with the use of multiple sets of hybridization primers and probes, each set labeled with a separate fluorescent reporter label.