Multiplex Nucleic Acid Detection Kit Using Temperature-Gradient Probes
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Solution Overview
Problem
Current methods for detecting multiple target nucleic acids, such as those involved in genetic screening, require lengthy and costly processes, including independent measurements and complex wavelength settings, often necessitating multiple reaction vessels and optical systems, which increase time and expenses.
Innovation Solution
A method utilizing a single reaction vessel with a mixed solution and a single label, employing specific primers and probes with distinct annealing and detection temperatures, allowing for simultaneous amplification and detection of multiple nucleic acids without the need for melting curve analysis, using QProbe, Eprobe, or TaqMan probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If independent measurement is performed for each target nucleic acid, then detection accuracy is improved, but measurement time and cost increase significantly
Solution Approach 1:
The patent combines multiple independent detection processes into a single multiplex PCR reaction. Multiple target nucleic acids are amplified simultaneously in one reaction vessel using multiple primer pairs, eliminating the need for separate measurement processes for each target. This merging approach maintains detection accuracy while significantly reducing measurement time and operational complexity.
Solution Approach 2:
The invention creates a universal detection system that can simultaneously detect multiple different target nucleic acids using a single reaction mixture. The system employs multiple probes with different fluorescent labels that can distinguish between different targets through wavelength differentiation, allowing one reaction system to perform multiple detection functions simultaneously.
2Adaptability or versatility
If different pilot dyes are used to distinguish multiple target nucleic acids, then detection capability is improved, but analyzer cost and operational complexity increase
Solution Approach 1:
The patent utilizes fluorescent probes with different emission wavelengths (different colors) to distinguish multiple target nucleic acids. Each probe is labeled with a fluorophore that emits at a characteristic wavelength, allowing the detection system to differentiate between targets based on the color of fluorescence emitted. This approach enhances detection capability while using standard multi-wavelength detection capabilities.
Solution Approach 2:
The invention differentiates between multiple targets by detecting fluorescence at different wavelengths. The system monitors fluorescence signals at multiple wavelength channels simultaneously, using wavelength as a distinguishing parameter to identify different target nucleic acids. This parameter-based differentiation improves versatility without requiring complex hardware modifications.
3Measurement precision
If melting curve analysis is performed to distinguish multiple targets, then detection accuracy is improved, but analysis time increases to 5-10 minutes
Solution Approach 1:
The patent incorporates detection information directly into the PCR amplification process itself. By using probes that emit fluorescence when bound to target sequences during amplification, the system obtains detection data simultaneously with amplification, rather than requiring a separate post-amplification melting curve analysis step. This preliminary detection approach maintains accuracy while eliminating the time-consuming separate analysis phase.
Solution Approach 2:
The invention enables continuous detection throughout the PCR process by monitoring fluorescence signals at each amplification cycle. The probe-based detection system provides real-time information about target presence during amplification, allowing continuous useful action rather than interrupting the process for separate analysis. This continuity maintains detection precision while significantly reducing total analysis time.
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 significantly reduces measurement time and costs by enabling the detection of multiple target nucleic acids using a single reaction vessel and label, while maintaining accurate differentiation through fluorescent signal changes.
Implementation Method 1
a first target's probe at the annealing temperature T1 specifically bonding with either of the first two single strands into which the first target nucleic acid has been dissociated
Implementation Method 2
the first target's probe including a first labeling substance changing first fluorescent signals thereof when the first target's probe specifically bonds with either of the first two single strands
Implementation Method 3
deoxyribonucleoside triphoshate at an elongation temperature T2 bonding by action of the DNA polymerase with both of the first two single strands into which the first target nucleic acid has been dissociated and the second two single strands into which the second target nucleic acid has been dissociated
Implementation Method 4
at a denaturation temperature TO, first double-stranded hydrogen bond of the first target nucleic acid being cut off to be dissociate into first two single strands
Data Source
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AI summary
Provided is a kit for detecting multiple target nucleic acids capable of simultaneously amplifying and detecting multiple genes by means of one reaction vessel containing one kind of reaction solution and one kind of labels. Solution may contain first target nucleic acid (10) and second target nucleic acid (20) each of which dissociates at denaturation temperature T0. The solution further contains: DNA polymerase (30); a first target's primer (13) at annealing temperature T1 bonding with first single strands derived from the first target nucleic acid; a second target's primer (23) at annealing temperature T1 bonding with second single strands derived from the second target nucleic acid; a first target's probe (15) at annealing temperature T1 bonding with the first single strands derived from the first target nucleic acid; and a second target's probe (25) at second target detection temperature T3 which is lower than the annealing temperature T1 and elongation temperature T2 bonding with the second single strands derived from the second target nucleic acid. A condition that: T0 is higher than T2; T2 is not lower than T1; and T1 is higher than T3 is satisfied.