Nucleic Acid Detection via Salt-Optimized PCR-Microarray Integration
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Solution Overview
Problem
Current nucleic acid detection methods, such as real-time PCR and microarray methods, face challenges in achieving high sensitivity and simultaneous detection of multiple target genes, necessitating the development of a more efficient detection method.
Innovation Solution
A nucleic acid detection method involving a probe-immobilized substrate with a nucleic acid probe complementary to the target sequence, a reaction liquid with a specific salt concentration, and a marker substance that produces a detectable signal upon binding with the amplification product, allowing for the determination of the target nucleic acid's presence and quantity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time PCR method is used for nucleic acid detection, then sensitivity is high and quantitative range is wide, but it cannot detect multiple target genes simultaneously
Solution Approach 1:
The patent combines real-time PCR amplification with microarray detection into a single integrated system. The PCR reaction is performed in the presence of immobilized probes on the microarray substrate, allowing simultaneous amplification and detection of multiple targets. This merging of two separate methods enables both high sensitivity (from PCR) and multi-target capability (from microarray).
Solution Approach 2:
The microarray substrate serves multiple functions: it acts as both the detection platform with immobilized probes and the reaction vessel for PCR amplification. The same substrate enables detection of multiple different target sequences simultaneously, providing universal applicability for various nucleic acid targets without requiring separate reactions for each target.
2Adaptability or versatility
If microarray method is used for nucleic acid detection, then multiple target genes can be detected simultaneously, but sensitivity and quantitative range are limited
Solution Approach 1:
The patent merges microarray probe immobilization with PCR amplification reagents into a single reaction system. The immobilized probes on the microarray substrate directly participate in the PCR reaction, enabling the amplification products to be detected with high sensitivity while maintaining the multi-target capability of the microarray platform.
3Reliability
If separate amplification and detection steps are used, then detection can be performed, but the process is complex and time-consuming
Solution Approach 1:
The patent combines the amplification step (PCR) and the detection step (hybridization) into a single simultaneous process. The immobilized probes on the microarray substrate serve as both primers for amplification and detectors for the amplified products, eliminating the need for separate steps and reducing overall process complexity.
Solution Approach 2:
The probes are pre-immobilized on the microarray substrate before the reaction begins, and the substrate is designed to support both amplification and detection functions from the outset. This preliminary preparation allows the system to perform multiple functions without requiring additional processing steps during the reaction.
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 enables high-sensitivity and high-precision detection of nucleic acids, capable of quantifying multiple targets simultaneously, improving upon existing techniques by optimizing salt concentrations for amplification and hybridization reactions.
Implementation Method 1
a polymerase which produces amplification product nucleic acid including the first sequence or its complementary sequence
Implementation Method 2
a nucleic acid probe one end of which is immobilized to the substrate, the nucleic acid probe including a sequence complementary to the first sequence
Implementation Method 3
a marker substance which produces a detectable signal, and detecting a signal from the marker substance
Data Source
AI summary
The nucleic acid detection method includes maintaining a reaction liquid containing the sample, a marker substance, polymerase, a primer set, and salt of a predetermined concentration in the reaction field which include a substrate and a probe, under an amplification condition, detecting a signal from the marker substance and determining existence and/or quantity of the target nucleic acid. The predetermined concentration is higher than the concentration in which the peak rate of the amplification reaction and the concentration with which the hybridization rate becomes higher than 0 au, whichever is higher, but not greater than the highest one of the concentrations with which the quantity of detection of the amplification product becomes a threshold value or more.