Multiplex Primer Extension for High-Throughput Pathogen RNA Detection

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Solution Overview

Problem

Next-generation sequencing (NGS) has not been successfully implemented for routine clinical pathogen diagnosis despite its advantages over traditional methods, such as unbiased protocols and ability to detect fastidious organisms, due to challenges in effectively detecting and diagnosing pathogens like SARS-CoV-2 in clinical settings.

Innovation Solution

A high-throughput method involving multiplex primer extension reaction for pathogen RNA detection, including purification, reverse transcription, barcode labeling, multiplex PCR, and sequencing, which allows for the identification of pathogen RNA in clinical specimens like nasopharyngeal swabs and FFPE tissue samples, using a combination of target-specific primers and template switching oligos to amplify and index the cDNA libraries for NGS analysis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If NGS is used for pathogen detection, then detection comprehensiveness and ability to detect fastidious organisms is improved, but implementation success for routine clinical diagnosis remains poor

Engineering Contradiction:

Improvedetection comprehensivenessVSAvoidimplementation success

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The method segments the NGS workflow into standardized modules: RNA extraction, reverse transcription with template switching, multiplex PCR amplification, library preparation, and sequencing. Each module is optimized and validated independently, allowing the complex pathogen detection process to be reliably implemented in clinical settings.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent performs preliminary actions by designing and validating the complete NGS pipeline before clinical implementation, including optimizing reverse transcription conditions, selecting multiplex PCR primer sets, and establishing quality control thresholds. This preliminary preparation ensures reliable implementation when the method is deployed for routine diagnosis.

Inventive Principle:

Principle #10Preliminary action

2Ease of operation

If traditional pathogen-specific protocols are used, then diagnostic simplicity is maintained, but detection of fastidious or non-culturable organisms is limited

Engineering Contradiction:

Improvediagnostic simplicityVSAvoiddetection capability

Core Design Contradiction:

Ease of operationVSAdaptability or versatility

Solution Approach 1:

The patent implements a universal NGS-based diagnostic platform that can detect any pathogen including fastidious and non-culturable organisms. The method uses universal RNA extraction, template switching for cDNA synthesis, and multiplex PCR with pathogen-specific primers, allowing a single protocol to serve multiple diagnostic purposes while maintaining operational simplicity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Adaptability or versatility

If NGS is applied to generate data across many disciplines, then data utility and diagnostic capability are improved, but process complexity and implementation difficulty increase

Engineering Contradiction:

Improvedata utilityVSAvoidprocess complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent optimizes critical parameters to balance data utility and process complexity: adjusting reverse transcription temperature and time, optimizing multiplex PCR cycle conditions, selecting appropriate library preparation protocols, and setting sequencing depth parameters. These parameter optimizations ensure high-quality pathogen detection data while keeping the workflow manageable for clinical implementation.

Inventive Principle:

Principle #35Parameter changes

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

This method enables sensitive and accurate detection of SARS-CoV-2 RNA, including mutations, across a wide range of viral loads, with high throughput and the ability to process multiple samples efficiently, overcoming previous limitations in pathogen detection and diagnosis.

Implementation Method 1

converting RNA into n cDNA preparations by reverse transcription

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

amplifying a plurality of targets of the pathogen in each of the m wells by multiplex PCR

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS10941453B1High throughput detection of pathogen RNA in clinical specimens

Publication Date: 2021.03.09 PARAGON GENOMICS INC

AI summary

Laboratory-based high throughput methods and compositions (e.g., kits) for the detection of pathogen mRNA, including in particular, for the detection of SARS-CoV-2 RNA from up to 85,000 samples in one Illumina NovaSeq sequencing run. These methods may include barcoding cDNA, indexing pools of libraries and intensive use of automatic liquid handling, providing a ready-to-sequence library mix. These methods and compositions may allow a very inexpensive per sample cost and an assay that may be performed in about one day or less.