THD Primer Real-Time Nucleic Acid Detection
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Solution Overview
Problem
Conventional methods for detecting target nucleic acid sequences are inefficient, time-consuming, and costly, often requiring complex primer designs and additional probes, which complicate the detection process and are not suitable for multiplex assays.
Innovation Solution
The development of target hybridization and detection primers (THD primers) with dual surveillance functions, incorporating a 5'-cleavage reaction and 3'-extension reaction using template-dependent nucleic acid polymerases, allowing for real-time detection without the need for additional probes or complex primer modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional detection methods are used, then detection can be performed, but the process is time-consuming and low-throughput
Solution Approach 1:
The patent combines the hybridization probe function and primer function into a single integrated molecule called THD primer. This merging eliminates the need for separate probes and primers, streamlining the detection process and enabling real-time monitoring without additional steps, thereby increasing throughput and reducing detection time.
Solution Approach 2:
The THD primer serves multiple functions simultaneously: it acts as a hybridization probe for target recognition, a primer for DNA synthesis initiation, and a template for forming hairpin structures that generate fluorescence signals. This multi-functionality consolidates multiple detection steps into one, significantly improving productivity and reducing time loss.
2Measurement precision
If additional probes and complex primer modifications are used, then detection sensitivity can be improved, but device complexity increases
Solution Approach 1:
The patent merges the probe sequence and primer sequence into a single THD primer molecule with a specific structure containing a 5' probe region, a central primer region, and a 3' tail region. This integration maintains high detection sensitivity through the probe's target-specific hybridization while eliminating the complexity of managing separate probes and primers, thus improving the sensitivity-to-complexity ratio.
3Reliability
If conventional post-PCR detection methods are used, then amplified products can be detected, but carryover contamination occurs
Solution Approach 1:
The patent implements real-time feedback monitoring of fluorescence signals during the PCR amplification process. The THD primer's hairpin structure generates fluorescence only when hybridized to the target sequence, providing continuous feedback that allows detection before the PCR products are complete, thereby preventing carryover contamination while maintaining detection accuracy.
4Reliability
If real-time detection methods are implemented, then contamination is avoided, but additional probes and complex systems are required
Solution Approach 1:
The THD primer is designed to perform multiple functions within a single molecule: target hybridization, primer annealing, and fluorescence signal generation through hairpin formation. This universality enables real-time contamination-free detection without requiring separate probes or complex additional components, thus maintaining reliability while minimizing system complexity.
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
The THD primer method enables efficient, real-time detection of target nucleic acid sequences with stronger and faster signal generation, simplifying the development of PCR assays and enabling multiplex detection without false positives.
Implementation Method 1
the THD primer is extended at its 3'-end by the polymerase activity of the template-dependent nucleic acid polymerase
Implementation Method 2
the label is released from the THD primer by the 5'-cleavage reaction, thereby generating a signal indicative of the presence of the target nucleic acid sequence
Implementation Method 3
The THD primer is hybridized with a target nucleic acid sequence
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence using a target hybridization and detection primer (THD primer). The present invention allows for both a target amplification and a signal amplification by introducing a label into a primer used in PCR reactions, ensuring a real-time target detection by PCR reaction by no use of complicated oligonucleotides. The present invention could completely be free from the troublesome matters and shortcomings associated with conventional real-time PCR methods. The present invention allows for successful real-time target detection by using only a labeled primer. This feature makes it possible that the present invention exhibits excellent real-time target detection in multiplex manner.