ID-Primers for Sample-Specific Viral Detection in Pooled Testing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current pooled testing methods for infectious diseases, such as COVID-19, face challenges including reduced sensitivity and selectivity due to sample dilution and cross-contamination, requiring retesting and being resource-intensive, especially in resource-poor regions.
Innovation Solution
The Uni-Pool method uses ID-Primers with unique sequences to tag and differentiate samples within a pool, allowing for sample-specific readouts through high-resolution melting curve analysis, eliminating the need for retesting and reducing assay time without compromising sensitivity or specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If pooled testing is used to reduce the number of tests, then testing capacity and cost efficiency are improved, but sensitivity is reduced due to sample dilution
Solution Approach 1:
The patent divides the pooled sample detection into multiple segments by assigning unique ID-Primers to each individual sample. Each ID-Primer contains a unique identifier sequence that allows the system to segment and track which specific samples in the pool tested positive, eliminating the need for retesting while maintaining sensitivity through sample-specific detection.
Solution Approach 2:
The patent changes the detection parameter by using high-resolution melting curve analysis to detect subtle differences in melting temperatures caused by the presence of different ID-Primer sequences. This allows the system to distinguish between samples from different individuals even when diluted in a pool, maintaining sensitivity while enabling pooled testing.
2Loss of time
If pooled testing is used to reduce the number of tests, then operation time and cost are saved, but selectivity is compromised due to higher probability of cross-contamination
Solution Approach 1:
The patent segments the detection process by using unique ID-Primers for each sample, allowing the system to identify which specific samples in the pool are positive. This eliminates cross-contamination concerns because each sample's result is independently tracked through its unique identifier, maintaining selectivity while enabling pooled testing.
Solution Approach 2:
The ID-Primer acts as an intermediary that links each individual sample to its unique identifier. This intermediary allows the system to trace positive results back to specific samples without direct cross-contamination between samples, as the unique sequence serves as a molecular barcode that distinguishes each sample's contribution to the pool.
3Productivity
If traditional pooled testing is used, then the number of tests is reduced, but retesting is required which increases overall time and resource usage
Solution Approach 1:
The patent performs preliminary action by incorporating unique ID-Primers into each sample before pooling. This preliminary tagging allows the system to identify positive samples directly from the pooled result without needing to retest individuals, as the unique identifiers are already present in the amplified products and can be distinguished through melting curve analysis.
Solution Approach 2:
The patent implements feedback by using the melting temperature information from the pooled PCR reaction to directly identify which specific samples are positive. The unique ID-Primer sequences produce distinct melting temperatures that provide immediate feedback about the status of each individual sample within the pool, eliminating the need for retesting.
4Measurement precision
If NGS-based massively parallel diagnostic assays are used to achieve sample-specific detection, then detection accuracy is improved, but cost and device complexity increase significantly
Solution Approach 1:
The patent uses inexpensive, simple ID-Primer sequences as disposable molecular barcodes instead of expensive NGS consumables. These short oligonucleotide sequences with unique identifiers can be easily synthesized and used in standard PCR machines, providing sample-specific detection accuracy at a fraction of the cost and complexity of NGS-based approaches.
Solution Approach 2:
The patent replaces the complex mechanical and computational system of NGS sequencing with a simpler biochemical approach using melting curve analysis. Instead of requiring expensive sequencing instruments and complex bioinformatics processing, the system uses the inherent thermal properties of DNA sequences to provide sample-specific identification through standard real-time PCR equipment.
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 efficient, rapid, and cost-effective large-scale screening by differentiating 2-50 samples in a single pool with minimal cross-reactivity, reducing the need for secondary testing and shortening turnaround times.
Implementation Method 1
a sample-specific readout by incorporating a sample-specific identifier strand of different sequences (i.e., with distinguishable Gibbs free energy and/or melting temperature) to the 5′ end of a primer complementary to the target nucleotide sequence
Implementation Method 2
Each sample outcome (positive or negative) in one pool can be obtained by high-resolution melting curve analysis (HRMCA) after qPCR
Data Source
AI summary
The subject invention pertains to methods for the analysis of pooled samples without the need of retesting through the use of oligonucleotide hybridization and target-specific amplification reactions. Specifically, a series of identifier oligonucleotides with different sequence compositions, each corresponding to a distinct sample, are combined into the target template of interest through nucleic acid synthesis. The aforementioned products are pooled together, and the pooled samples are amplified and detected using the probe-based hybridization assay or a size separation module to identify if any of the pool of samples test positive, as well as simultaneously identifying which sample is positive for the targeted sequence.