Barcoded PCR Sequencing Assay for High-Throughput COVID Testing
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Solution Overview
Problem
Existing diagnostic infrastructure for pathogens like SARS-CoV-2 is inadequate for sudden, large-scale testing demands, requiring significant equipment and labor, and existing test protocols are not adaptable to mutating viruses.
Innovation Solution
A method and system using molecular barcoding and next-generation sequencing for parallel detection of SARS-CoV-2, involving reverse transcription, pooled PCR amplification, and automated processes to reduce equipment needs and increase throughput.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional diagnostic testing infrastructure is used to handle large-scale testing demands, then testing capacity can be increased, but equipment requirements and physical footprint increase significantly
Solution Approach 1:
The patent combines multiple individual diagnostic tests into a single pooled testing workflow. Multiple patient samples are reverse transcribed and pooled together, then subjected to a single PCR reaction with barcoded primers. This merging approach allows thousands of samples to be processed using minimal equipment (one RT machine, one PCR machine, one sequencer) rather than requiring separate equipment for each test, thereby increasing testing capacity without proportionally increasing device complexity.
Solution Approach 2:
The patent employs universal reagents and protocols that can handle diverse samples and target multiple viral variants simultaneously. The barcoded primer system is designed to work with different SARS-CoV-2 variants, and the sequencing platform can process various nucleic acid types. This multi-functionality allows a single testing infrastructure to serve multiple purposes and handle large volumes of samples without requiring specialized equipment for each test type.
2Measurement precision
If traditional diagnostic testing is performed on multiple samples individually, then testing accuracy is maintained, but processing time and labor requirements increase
Solution Approach 1:
Multiple individual testing workflows are merged into a single pooled workflow. Instead of performing separate RT-PCR reactions for each sample, the patent combines multiple samples in one pooling step, followed by a single PCR reaction with barcoded primers. The sequencing step simultaneously reads all barcoded products. This merging reduces processing time from days to hours while maintaining accuracy through barcode-based sample identification and quantification.
Solution Approach 2:
The patent uses molecular barcodes as informational copies that allow simultaneous tracking of multiple samples. Each sample receives a unique barcode during reverse transcription, creating a molecular copy of the sample identity that persists through pooling and amplification. This copying mechanism enables the system to process many samples in parallel while maintaining the ability to individually identify and quantify results for each original sample, thus reducing processing time without sacrificing accuracy.
3Reliability
If fixed test protocols are used for pathogen detection, then test reliability is maintained, but adaptability to viral mutations is reduced
Solution Approach 1:
The patent implements a dynamic primer design system where barcoded primers can be updated to target new viral variants. Instead of fixed protocols, the system allows for rapid redesign of the barcode sequences and primer binding regions based on emerging variant data. The high-throughput sequencing platform can accommodate these changes without requiring fundamental protocol restructuring, enabling the system to adapt to mutations while maintaining reliability through consistent core workflows.
Solution Approach 2:
The patent changes key parameters of the testing system - specifically the barcode sequences and primer designs - to adapt to viral mutations. By modifying these parameters rather than the entire testing protocol, the system maintains reliability of the core RT-PCR-seq workflow while adapting to new variants. The modular nature of the barcode system allows parameter changes to be implemented rapidly in response to emerging viral variants.
4Productivity
If high-throughput testing is implemented using traditional methods, then testing volume increases, but cost per test and resource requirements increase
Solution Approach 1:
The patent merges resource requirements by pooling multiple samples together for shared processing steps. Instead of each sample consuming dedicated reagents and equipment time, multiple samples share the same PCR reaction mix, the same thermal cycler, and the same sequencing run. This merging dramatically reduces the quantity of substances (reagents, enzymes, buffers) needed per test while maintaining high testing volume through parallel processing of pooled samples.
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 high-throughput testing with a reduced physical footprint, capable of processing thousands of samples daily while maintaining sensitivity and adaptability to viral mutations.
Implementation Method 1
performing reverse transcription using the RT reaction mixtures to generate a RT reaction product comprising cDNA with a sample-specific barcode
Implementation Method 2
performing PCR using the PCR reaction mixture to generate amplified cDNA comprising both a sample-specific barcode and a pool-specific barcode
Data Source
AI summary
The present disclosure provides methods and systems for the massively-parallel detection of pathogens, such as SARS-CoV-2 virus, in a set of multiple samples via PCR test. Various implementations may provide for barcode/primer sequences that are designed to allow for a large number of samples and/or multiple pathogens to be analyzed in a single test. Included within the scope hereof are methods and systems for performing tests of multiple samples at once, via a one-pot test protocol, as well as methods and systems for designing test parameters (such as barcode/primer sequences) in a manner that allows for parallel testing.