Two-Step Nucleic Acid Genotyping With Multiplex Barcoding
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Solution Overview
Problem
Existing methods for nucleic acid sequence determination and classification, particularly in the context of antibiotic resistance detection, are slow and lack the capacity for accurate multiplexing, hindering effective treatment and infection control.
Innovation Solution
A two-step primer-dependent amplification process involving selective amplification and barcoding, using sets of selection and amplification primers in controlled concentrations to enable high multiplexing and specific amplification of target nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional cultivation-based methods are used for antibiotic susceptibility testing, then accuracy of resistance determination is improved, but testing time increases to at least 24 hours
Solution Approach 1:
The method performs preliminary amplification of target nucleic acid sequences (including antibiotic resistance genes) before final detection. By pre-amplifying the genetic material in the first PCR step, the system prepares sufficient target material for subsequent multiplexing and genotyping, enabling rapid results without sacrificing accuracy
Solution Approach 2:
The testing process is divided into distinct segments: initial amplification of target sequences, second amplification with genotype-specific primers, and detection phases. This segmentation allows each step to be optimized independently, achieving both speed and accuracy
2Loss of time
If conventional molecular methods like real-time PCR are used for rapid detection, then testing time is reduced, but multiplexing capacity is limited preventing complete resistance profile determination
Solution Approach 1:
The PCR system is designed with universal components that can detect multiple antibiotic resistance genes simultaneously. By using universal amplification conditions combined with genotype-specific primers in the second step, the method achieves high multiplexing capacity to determine complete resistance profiles while maintaining rapid testing
Solution Approach 2:
The method adds a temporal dimension by performing amplification in two sequential steps rather than attempting to detect all targets simultaneously in a single step. This dimensional approach allows complex multiplexing to be achieved through time-separated operations
3Adaptability or versatility
If multiple antibiotic resistance genes are detected simultaneously through multiplexing, then complete resistance profile is determined, but assay complexity increases
Solution Approach 1:
The complex multiplexing assay is segmented into two distinct PCR steps. The first step amplifies all target sequences universally, while the second step uses genotype-specific primers for differentiation. This segmentation reduces the complexity of each individual step while maintaining the overall multiplexing capability
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
Enables rapid and reliable amplification and genotyping of nucleic acid sequences, facilitating efficient detection of antibiotic resistance and improving treatment outcomes by allowing simultaneous processing of multiple sequences in a single reaction.
Implementation Method 1
The first amplification step is a selective amplification of the target nucleic acid sequence using a set of selection primers and a first set of amplification primers in a primer-dependent enzymatic reaction to yield an amplicon
Data Source
Figure 1~2
Figure 3A~3D
Figure 3E~3H
AI summary
An amplification of a target nucleic acid sequence (1) is a primer-dependent amplification involving two amplification step. The first amplification step is a selective amplification of the target nucleic acid sequence (1) using a set of selection primers (10, 20) and a first set of amplification primers (30, 40) in a primer-dependent enzymatic reaction to yield an amplicon (4). The second amplification step is an amplification and barcoding of the amplicon (4) using a barcoding primer (50) and a second set of amplification primers (30, 60; 40, 70) in a primer-dependent enzymatic reaction to yield a barcoded amplicon (6). A high degree of multiplexing is achieved in the amplification by having selection primers (10, 20) and barcoding primers (50) in comparatively lower concentrations as compared to amplification primers (30, 60; 40, 70).