Stem-and-loop nucleic acid detection for isothermal amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid detection methods, such as PCR and LAMP, face challenges including complex temperature control, low sensitivity due to double-stranded product interference, and inability to detect multiple target genes simultaneously, with existing LAMP methods lacking confirmation of unintended amplification products and requiring complex enzyme handling.
Innovation Solution
A method involving stem-and-loop structured nucleic acids with complementary sequences at terminals and a target sequence in between, using probe nucleic acids immobilized on a solid substrate for specific hybridization and detection, allowing for simultaneous analysis of multiple targets without denaturation procedures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR method is used for nucleic acid amplification, then amplification can be achieved, but complex temperature control device is required and reaction time exceeds two hours
Solution Approach 1:
The patent changes the temperature parameter from variable (PCR) to constant isothermal condition (65°C), eliminating the need for complex temperature cycling devices while maintaining amplification efficiency and reducing reaction time to under two hours
Solution Approach 2:
The patent replaces the mechanical temperature cycling system with a simple heating system maintained at constant 65°C, substituting complex thermal control mechanics with a straightforward isothermal heating approach
2Productivity
If double-stranded products are generated in PCR amplification, then amplification efficiency is maintained, but hybridization efficiency decreases due to complementary strands working as competitors for probes
Solution Approach 1:
The patent extracts and removes the complementary strand from the double-stranded PCR product through digestion or separation, leaving only the target single-stranded sequence to hybridize with probes, thereby eliminating competition and improving hybridization efficiency
Solution Approach 2:
The patent introduces enzymes or separation methods as intermediaries to process the PCR products, converting double-stranded DNA into single-stranded form suitable for probe hybridization without directly altering the probe or target sequences
3Productivity
If LAMP method is used for amplification, then amplification speed and sensitivity are improved, but inability to detect multiple target genes simultaneously and confirmation of unintended products remains a problem
Solution Approach 1:
The patent segments the detection process into multiple independent probe channels, each targeting different gene sequences, allowing simultaneous detection of multiple targets in a single LAMP reaction by measuring hybridization signals from different probe sets
Solution Approach 2:
The patent creates a universal detection platform where the same LAMP amplification system can detect multiple different target genes simultaneously through the use of multiple specific probes, making the system multi-functional rather than single-purpose
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 enhances detection sensitivity and specificity, enabling rapid identification of bacterial species and genetic mutations with improved hybridization efficiency and the ability to detect single nucleotide polymorphisms without steric hindrance, facilitating quicker and more accurate clinical diagnostics.
Implementation Method 1
reacting the nucleic acid for measurement with the probe nucleic acid to specifically hybridize the target sequence portion of the nucleic acid for measurement to the probe nucleic acid
Data Source
AI summary
A method of detecting a target nucleic acid sequence comprising providing a stem-and-loop structured nucleic acid for measurement wherein the nucleic acid comprises complementary sequence portions located at both terminals and a target sequence portion therebetween as well as a double-stranded portion formed by hybridization of the complementary sequence portions located at both terminals and a remaining looped single-stranded portion, providing a probe nucleic acid having a sequence complementary to the target sequence portion wherein one end of the probe nucleic acid being immobilized to a solid substrate surface, reacting the nucleic acid for measurement with the probe nucleic acid to specifically hybridize the target sequence portion of the nucleic acid for measurement to the probe nucleic acid, and detecting presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid.