Multiplex Analysis System Using Universal Tail Sequences for Primer Compatibility
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Solution Overview
Problem
Current diagnostic methods for disease agents, particularly in bioterrorism scenarios, face challenges in rapid and accurate identification due to nonspecific clinical signs, leading to potential misdiagnosis and ineffective containment, with existing PCR tests lacking sensitivity and requiring complex optimization procedures for multiplex reactions.
Innovation Solution
The Multiplex Analysis System (MAS) employs a rapid multiplex amplification and detection method using Tem-PCR, which simultaneously amplifies and detects nucleic acid sequences from disease agents and secondary agents, allowing for differential diagnosis in as little as 3 hours without extensive optimization, using a combination of nucleic acid isolation, multiplex amplification, and specific primer strategies to reduce incompatibility and enhance sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex PCR is used to detect multiple disease agents simultaneously, then diagnostic efficiency and productivity are improved, but primer incompatibility and amplification interference increase, reducing measurement precision
Solution Approach 1:
A universal tail sequence is introduced as an intermediary element on the 5' end of each pathogen-specific primer. This tail contains a common binding site that allows all pathogen-specific primers to be recognized by a single reporter probe, thereby mediating the interaction between multiple primers and the detection system. This resolves primer incompatibility by providing a standardized interface that eliminates amplification interference while maintaining the ability to detect multiple disease agents simultaneously through a unified detection mechanism
2Measurement precision
If conventional PCR optimization procedures are performed for multiplex reactions, then amplification precision is improved, but time consumption and device complexity increase
Solution Approach 1:
The invention changes the fundamental parameters of the PCR system by introducing a universal tail sequence and a single reporter probe that recognizes this tail. This parameter change transforms the optimization requirements from needing multiple pathogen-specific probes with different binding conditions to using one universal probe that binds to the common tail sequence. Consequently, the complex multi-parameter optimization process is replaced by a standardized protocol that achieves high amplification precision without extensive time-consuming optimization
Solution Approach 2:
The universal tail sequence serves multiple functions simultaneously: it enables specific amplification of each pathogen's DNA, provides a common binding site for the reporter probe, and facilitates detection of multiple pathogens in a single reaction. This multi-functionality eliminates the need for separate optimization of multiple probe-primer interactions, thereby reducing both time consumption and device complexity while maintaining high amplification precision
3Reliability
If existing PCR tests are used to detect disease agents, then specificity is improved, but sensitivity is reduced due to inability to detect low viral loads
Solution Approach 1:
The invention merges the detection of multiple pathogens into a single multiplex PCR reaction by using pathogen-specific primers that amplify unique regions of each pathogen's DNA, all containing a universal tail sequence. This merging allows simultaneous amplification of multiple targets in one reaction tube, increasing sensitivity by detecting low viral loads of multiple pathogens together while maintaining specificity through pathogen-specific primer design and a single universal reporter probe that distinguishes between different pathogens
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
MAS enables rapid and accurate diagnosis of disease agents and secondary agents, reducing the risk of misdiagnosis, improving containment, and allowing for high-throughput analysis without the need for complex optimization procedures, thereby enhancing public health responses to bioterrorism threats.
Implementation Method 1
molecular tests generally use any one of a number of variations on the polymerase chain reaction (PCR)
Implementation Method 2
primer based multiplex amplification of nucleic acids employing primer binding tags
Data Source
AI summary
Disclosed is a novel method for diagnosis or differential diagnosis of disease agents and secondary disease agents. The method disclosed uses a novel amplification strategy termed TemPCR to allow sensitive and specific amplification of target sequences from any disease agents and/or secondary disease agent whose nucleic acid sequence is known. The TemPCR method utilizes at least one set of target enrichment primers specific for the disease agent or secondary disease agent to be detected (present at a low concentration) and at least one pair of shared target amplification primers (present at high concentrations). At least one pair of said target enrichment primers comprises a binding sequence for the target amplification primers. Therefore, the use of the TemPCR method allows multiplex amplification reactions to be carried out without the need for empirical optimization of the multiplex amplification parameters. Methods for nucleic acid isolation and the detection of the target sequences are also disclosed for use with the TemPCR method.