RT-qPCR Primer Segmentation for SARS-CoV-2 Detection

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Solution Overview

Problem

Current SARS-CoV-2 diagnostic assays face issues with sensitivity, specificity, and workflow efficiency, particularly due to uneven amplification, false negatives, and cross-reactivity, which hinder accurate detection and differentiation from seasonal influenza viruses.

Innovation Solution

The development of improved RT-qPCR methods involving simultaneous amplification with specific primer nucleotide sequences for SARS-CoV-2 RdRP, E gene, and human RNase P, along with dual probes and internal controls, enhances sensitivity and specificity, and allows for differentiation from influenza A and B viruses, while also being room temperature-stable for reduced shipping and storage needs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If multiplex PCR methods are used to detect multiple targets simultaneously, then productivity and workflow efficiency are improved, but false positives and false negatives increase due to uneven amplification and primer dimer formation

Engineering Contradiction:

Improveworkflow efficiencyVSAvoiddetection accuracy

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent divides the multiplex PCR detection into separate singleplex PCR reactions for each target (SARS-CoV-2 RdRP, E gene, and human RNase P). This segmentation eliminates interference between primers and probes, preventing false positives and false negatives while maintaining the ability to detect multiple targets through sequential testing

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces an internal control (human RNase P) that acts as an intermediary to monitor the quality of RNA extraction and PCR reaction conditions. This internal control helps identify false negatives by confirming that the PCR system is functioning properly, thereby improving detection reliability

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If multiple primer sets are used in simultaneous amplification, then sensitivity for detecting different viral targets is improved, but specificity decreases due to cross-reactivity and preferential amplification

Engineering Contradiction:

Improvedetection sensitivityVSAvoidamplification specificity

Core Design Contradiction:

Measurement precisionVSManufacturing precision

Solution Approach 1:

The patent separates the detection of different viral targets into distinct PCR reactions with dedicated primer sets. Each primer set is optimized for its specific target without interference from other primers, eliminating cross-reactivity and preferential amplification while maintaining high sensitivity for each individual target

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent optimizes each primer set and probe combination specifically for its target sequence, ensuring local optimality for each detection reaction. This includes adjusting annealing temperatures, primer concentrations, and probe designs to maximize specificity for each individual target while maintaining high sensitivity

Inventive Principle:

Principle #3Local quality

3Measurement precision

If complex diagnostic protocols with multiple steps are implemented, then measurement precision for SARS-CoV-2 detection is improved, but loss of time in diagnostic workflow increases

Engineering Contradiction:

Improvedetection accuracyVSAvoiddiagnostic time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent combines multiple detection objectives into a unified diagnostic approach using the same RNA extraction protocol for all targets. By merging the sample preparation step and using standardized PCR conditions across different reactions, the patent reduces redundant steps while maintaining high detection accuracy for SARS-CoV-2 and differentiation from influenza viruses

Inventive Principle:

Principle #5Merging (Combining)

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

The improved methods provide enhanced sensitivity and specificity for SARS-CoV-2 detection, streamline diagnostic workflows, and enable effective differentiation from seasonal influenza, facilitating more accurate clinical and public health interventions.

Implementation Method 1

All of them are based on nucleic acid amplification in order to detect 2019-nCoV

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

The protocols published by WHO use different gene targets of 2019-nCoV... All these protocols apply quantitative/real-time RT-PCR assays

Methodology Applied

Scientific EffectPCR amplification:

Implementation Method 3

The present invention relates to a PCR-method comprising conducting a simultaneous amplification step with at least primer nucleotide sequences for SARS-CoV-2 RdRP gene

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS20230175081A1Means and methods for detecting novel coronavirus (SARS-cov-2)

Publication Date: 2023.06.08 MULTIPLEXDX SRO

AI summary

The present invention relates to a PCR-method comprising conducting a simultaneous amplification step with at least primer nucleotide sequences for SARS-CoV-2 RdRP gene, at least primer nucleotide sequences for SARS-CoV-2 E gene and/or at least primer nucleotide sequences for human RNase P gene. Said PCR-method may further comprise a conducting an amplification step, preferably a simultaneous amplification step, with at least primer nucleotide sequences for a unique spike RNA. Also provided is a kit comprising primers and optionally probes to carry out the PCR-methods of the invention.