Single Probe Nucleic Acid Detection via Multi-Temperature Discrimination
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Solution Overview
Problem
Current nucleic acid detection methods struggle to differentiate between multiple target sequences using a single probe, especially when these sequences have identical probe hybridization sites, which limits their ability to accurately identify polymorphic variants such as SCCmec types in Staphylococcus aureus, including MRSA and MSSA, due to reliance on different probes or non-identical hybridization sites.
Innovation Solution
Employing multiple temperature end-point signal probe detection in conjunction with asymmetric PCR amplification methods, such as LATE-PCR, to generate temperature-dependent signals that distinguish between target amplicons with identical probe hybridization sequences by exploiting secondary structure differences within the amplicons, allowing for the identification of polymorphic variants using a single labeled probe.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single probe is used for nucleic acid detection, then the simplicity and cost-effectiveness of the assay is improved, but the ability to differentiate between multiple target sequences with identical hybridization sites deteriorates
Solution Approach 1:
The patent introduces temperature as an additional dimension for discrimination. By detecting probe hybridization signals at multiple temperatures (e.g., during PCR annealing and extension phases), the system can distinguish between target sequences that have identical hybridization sites but differ in their downstream sequences. The temperature-dependent signal changes provide an extra degree of freedom for differentiation without adding multiple probes.
Solution Approach 2:
The patent utilizes changes in thermal parameters (temperature) to achieve sequence discrimination. By monitoring signal intensity or fluorescence at different temperatures, the assay exploits the temperature-dependent stability of probe-target hybrids. Sequences with different downstream regions create different thermal signatures, allowing discrimination through parameter variation rather than probe variation.
2Measurement precision
If multiple probes are used to differentiate target sequences, then the discrimination capability is improved, but the assay complexity and cost increase
Solution Approach 1:
The patent makes a single probe multi-functional by enabling it to detect multiple target sequences through temperature-dependent signal analysis. Instead of requiring separate probes for each target, the same probe can distinguish between sequences by monitoring signal characteristics at different temperatures, thereby reducing the number of probes needed while maintaining comprehensive detection capability.
Solution Approach 2:
The patent adds temperature as an additional detection dimension for a single probe. By measuring signal intensity at multiple temperatures (e.g., during PCR cycles), the system creates a thermal signature profile that enables differentiation between multiple targets using one probe, effectively multiplying the probe's functionality without physically replicating the probe.
3Ease of operation
If traditional PCR amplification is used, then the simplicity of the method is maintained, but the ability to detect polymorphic variants with identical hybridization sites is limited
Solution Approach 1:
The patent introduces temperature as an additional detection dimension during PCR. By monitoring probe signal intensity at multiple temperatures throughout the PCR process, the system can detect polymorphic variants that have identical hybridization sites but differ in their downstream sequences. The temperature-dependent signal variations provide a new degree of freedom for variant detection without complicating the basic PCR operation.
Solution Approach 2:
The patent implements feedback by continuously monitoring temperature-dependent signal changes during PCR cycles. The system uses real-time signal intensity measurements at different temperatures to provide feedback about the presence of specific polymorphic variants, allowing for dynamic detection and identification of variants as they are amplified.
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 enables the accurate discrimination of multiple target sequences with identical probe hybridization sites by generating distinct temperature/temperature signal ratios, effectively identifying polymorphic variants like SCCmec types in Staphylococcus aureus, improving the diagnostic capability for methicillin-resistant and susceptible strains.
Implementation Method 1
exposing the combined sample to multiple temperatures that allow the labeled probe to hybridize to the probe hybridization sequence and produce temperature-dependent signals
Implementation Method 2
by exploiting secondary structure differences within the amplicons, allowing for the identification of polymorphic variants using a single labeled probe
Data Source
AI summary
Provided herein are methods, kits, and compositions related to nucleic acid detection assays that allow discrimination of multiple target sequences with a single probe. In particular, provided herein are methods kits, and compositions that include single-probe target sequence discrimination where different target amplicons may have identical probe hybridization sequences by employing multiple temperature end-point signal probe detection. Also provided herein are methods, kits, and compositions for distinguishing between two or more target amplicons using multiple-temperature end-point probe detection. In certain embodiments, asymmetric PCR amplification methods are employed (e.g., LATE-PCR amplification).