cRASL-seq Multiplexed RNA Detection via Multi-Partite Probes
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Solution Overview
Problem
Current nucleic acid tests for SARS-COV-2 have limitations such as labor-intensive RNA extraction, high costs, low throughput, and inability to provide strain or clade-level information, which hinders effective surveillance and diagnosis during pandemics.
Innovation Solution
The development of capture RASL-seq (cRASL-seq) technology, which enables direct analysis of pathogen-associated RNA without nucleic acid extraction or reverse transcription, allowing for highly sensitive and multiplexed detection of pathogens, including SARS-COV-2, using multi-partite probes and next-generation DNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional RT-PCR is used for SARS-COV-2 detection, then cost is reduced, but labor intensity and time consumption increase due to separate RNA extraction and amplification steps
Solution Approach 1:
The patent combines RNA extraction, reverse transcription, and PCR amplification into a single cartridge-based reaction chamber. The multi-partite probe system (capture probe, acceptor probe, donor probe) enables all these steps to occur simultaneously in one automated workflow, eliminating sequential manual operations and reducing total testing time while maintaining cost-effectiveness.
Solution Approach 2:
The cartridge system serves multiple functions: it performs RNA extraction, reverse transcription, and amplification in a single integrated platform. The multi-partite probes provide universal applicability across different viral targets, allowing the same system to detect various pathogens without requiring separate extraction protocols.
2Ease of operation
If cartridge-based nucleic acid tests are used, then sample preparation is minimized and results are rapid, but scalability is limited due to production constraints and low throughput
Solution Approach 1:
The cartridge system is designed for automated operation with minimal manual intervention. The integrated reagents and probes within the cartridge perform all necessary functions automatically once the sample is loaded, enabling high-throughput processing without proportionally increasing labor requirements and facilitating scalable deployment.
3Measurement precision
If most current testing platforms are used, then single pathogen detection is achieved, but strain or clade-level information is not provided
Solution Approach 1:
The detection system is segmented into multiple independent probe sets, each targeting specific viral regions or variants. This segmentation allows simultaneous detection of different pathogens and strains within the same sample, providing both identification and characterization information without requiring separate tests.
4Adaptability or versatility
If multiplexed PCR platforms are used for infection diagnosis, then detection capability is improved, but cost and sensitivity are compromised along with complicated informatics
Solution Approach 1:
The system uses DNA copies of the viral RNA target for detection rather than requiring direct RNA analysis. The multi-partite probe system creates ligated DNA products that can be amplified and detected through standard PCR methods, reducing costs while maintaining multiplexing capabilities and simplifying data interpretation compared to direct RNA sequencing approaches.
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
cRASL-seq provides a scalable, cost-effective, and automated method for detecting pathogens and genotyping, enabling enhanced characterization of transmission chains and viral clades, with the potential to mitigate pandemics and prevent future crises.
Implementation Method 1
RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq)
Implementation Method 2
ligating the acceptor and donor probes to form a target RNA proxy
Data Source
AI summary
The present invention relates to the field of ribonucleic acid (RNA). More specifically, the present invention provides compositions and methods for highly multiplexed detection of pathogen-associated RNA. In a specific embodiment, a method for forming a target ribonucleic acid (RNA) proxy in a sample comprises the steps of (a) contacting a sample with one or more multi-partite probes that hybridize to a target RNA, wherein the one or more multi-partite probes comprise (i) a target capture probe, (ii) a 3′acceptor probe and (iii) a 5′ phosphorylated donor probe; (b) incubating the sample of step (a) under conditions that allow hybridization of the one or more multi-partite probes to target RNA present in the sample; (c) immobilizing the target capture probes on a solid support; (d) washing away unbound multi-partite probes; and € ligating the acceptor probes and donor probes to form a target RNA proxy.