Nucleic Acid Analysis Using Staggered Barcode Primers for Pooled Testing
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Solution Overview
Problem
Current nucleic acid testing methods for large-scale COVID-19 detection face challenges in increasing testing capacity, reducing costs, and maintaining sensitivity, particularly in multiplex assays using pooled RNA extracts.
Innovation Solution
A method involving primer extension reactions with staggered primers to identify samples in an array of subsets, followed by combining subsets for further amplification and sequencing, using unique identifier sequences to associate nucleic acid sequences with specific samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplexing is used to increase testing capacity, then the number of samples that can be tested increases, but the sensitivity of the assay decreases and the limit of detection increases
Solution Approach 1:
The invention segments the testing process into multiple independent barcode extension reactions, each targeting a specific viral sequence. By dividing the multiplex assay into separate barcode extension steps for different viruses, each reaction maintains high sensitivity while the overall system achieves high throughput. The segmentation allows individual optimization of each viral target without compromising detection limits.
Solution Approach 2:
The invention adds a temporal dimension to the multiplexing strategy by performing barcode extensions sequentially rather than simultaneously. This dimensional change allows multiple viral targets to be detected in a single sample while maintaining the sensitivity of individual assays, as each barcode extension reaction occurs in a dedicated time window without competitive interference from other targets.
2Productivity
If pooled RNA extracts are used for large scale testing, then testing capacity increases, but retesting is needed to identify individual positive samples
Solution Approach 1:
The invention performs preliminary barcode extension reactions that attach sample-specific identifiers to viral sequences before pooling. This preliminary action embeds the sample identity information directly into the nucleic acid molecules, eliminating the need for subsequent retesting to trace positive results back to individual samples. The barcode information is established in advance and preserved through the pooling process.
Solution Approach 2:
The sequencing step provides feedback that directly links detected viral sequences to their source samples through the embedded barcodes. This feedback mechanism allows immediate identification of which individual samples tested positive without requiring additional retesting, as the sample identity information is recovered directly from the sequencing data.
3Loss of information
If sample-derived DNA is barcoded to identify individual samples in multiplex approach, then sample identification is achieved, but the sensitivity of the assay is reduced
Solution Approach 1:
The invention applies local quality by placing barcode sequences in specific locations within the amplicon structure, rather than throughout the entire sequence. The barcodes are positioned in non-critical regions that do not interfere with primer binding or viral sequence detection, thereby maintaining assay sensitivity while achieving sample identification. Each local region of the amplicon serves its specific function without compromising others.
Solution Approach 2:
The invention uses the barcode sequence as an intermediary element that connects sample identity information to the viral sequence data. The barcode acts as a mediator that carries sample identification information without interfering with the detection of viral presence, allowing both functions to coexist by operating at different levels of the assay hierarchy.
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
Enhances testing capacity and sensitivity while reducing costs by accurately identifying individual samples within pooled nucleic acid tests, even with varying initial concentrations.
Implementation Method 1
a pair of primers comprises a forward and a reverse primer, wherein the forward and reverse primers both comprise in 5' to 3' direction an adaptor sequence, a sample identifier sequence and a binding sequence for hybridization to the analyte nucleic acids, respectively
Implementation Method 2
amplifying nucleic acids by a primer extension reaction using at least one pair of primers hybridized to the analyte nucleic acids
Data Source
Figure 1A~1E
Figure 1
Figure 2A~2E
AI summary
The present invention provides a method for detecting a nucleic acid of interest in a plurality of samples, comprising the steps of a) providing analyte nucleic acids from a plurality of samples in separate containers for each sample, wherein the containers are arranged into an array of subsets, wherein the array comprises two or more subsets; b) amplifying nucleic acids by a primer extension reaction using at least one pair of primers hybridized to the analyte nucleic acids, wherein a pair of primers comprises a forward and a reverse primer, wherein the forward and reverse primers both comprise an adaptor sequence, an sample identifier sequence and a binding sequence for hybridization to the analyte nucleic acids, respectively; c) combining the amplified nucleic acids of step b) of containers of two or more subsets to an array of combined containers, wherein containers of one subset, but not of another subset, are combined to a combined container; d) amplifying nucleic acids by a primer extension reaction using at least one pair of further primers hybridized to the amplified nucleic acids of combined containers of step c), wherein a pair of further primers comprises a further forward and a further reverse primer, wherein the further forward and further reverse primers both comprise a subset identifier sequence and a sequence for hybridization to the adaptor sequence; e) determining the sequences of the amplified nucleic acids of step d); f) assigning a determined sequence of a nucleic acid of interest of step e) to a sample through association to a subset and container with the subset identifier sequences and the sample identifier sequences. Further provided are sets of primers for such a method.