IQ-4 Quenched Oligonucleotide Probes for N3 Gene Detection
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Solution Overview
Problem
The existing RT-qPCR diagnostic panels for detecting 2019-nCoV are limited by the lack of detection capability for the N3 gene, leading to inconclusive test results and strain on resources due to the lower detection limit of the BHQ-1 and BHQ-2 quenchers, which are in short supply during high demand periods.
Innovation Solution
A kit comprising oligonucleotide probes modified with an IQ-4-based quencher that can detect multiple fragments of the 2019-nCoV nucleocapsid (N) gene, including N1, N2, and N3, using multiplexing RT-qPCR technology, enhancing detection efficiency and reducing signal/noise ratio.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If BHQ-1 or BHQ-2 quenchers are used in RT-qPCR probes, then fluorescence quenching efficiency is improved, but detection sensitivity for N3 gene deteriorates due to lower detection limit
Solution Approach 1:
The patent changes the chemical parameter of the quencher from BHQ-1/BHQ-2 to IQ-4, which has different fluorescence quenching properties. This parameter change enables the probe to detect N3 gene with improved sensitivity while maintaining effective fluorescence quenching, resolving the contradiction between quenching efficiency and detection sensitivity.
2Device complexity
If CDC 2019-nCoV RT-qPCR Diagnostic Panel targets only N1 and N2 genes, then test complexity is reduced, but detection reliability deteriorates due to inconclusive results when N3 is not detected
Solution Approach 1:
The patent applies multi-functionality by designing a single RT-qPCR diagnostic panel that can detect multiple viral genes (N1, N2, and N3) simultaneously using the same probe system with IQ-4 quencher. This allows the test to provide comprehensive detection coverage while maintaining operational simplicity, resolving the contradiction between test complexity and detection reliability.
3Quantity of substance
If BHQ-1 and BHQ-2 supplies are strained during high demand periods, then manufacturing availability deteriorates, but detection performance remains the same
Solution Approach 1:
The patent adopts IQ-4 quencher as an alternative to the depleted BHQ-1/BHQ-2 quenchers. This substitution enables continued production of RT-qPCR diagnostic panels during periods of high demand when original quencher supplies are strained, resolving the contradiction between supply availability and production capacity.
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 IQ-4-containing probes enable detection of all three nucleocapsid gene fragments at low levels, providing more accurate and certain test results with improved detection efficiency compared to BHQ-1 and BHQ-2, and maintaining a low signal/noise ratio.
Implementation Method 1
Each target-specific RT-qPCR probe comprises a sequence targeting a specific sequence in the viral RNA genome, a fluorophore attached to one end of the probe sequence and a quencher attached to the other end
Implementation Method 2
Close proximity of the quencher to the fluorophore attenuates the fluorescence of the fluorophore
Implementation Method 3
The amount of fluorescence at the end of each PCR cycle can be detected and registered by a qPCR instrument, and corresponds to the amount of cDNA and, therefore, RNA containing the targeted region
Data Source
AI summary
Provided herein are oligonucleotide probes for detecting 2019 novel coronavirus (2019-nCoV). The probes are modified at their 5′ ends with a fluorophore (e.g., fluorescein), and are also modified (e.g., at their 3′ ends) with a moiety capable of quenching fluorescence from the fluorophore. The moiety is based on the IQ-4 or IQ-2 quencher. Also provided are kits including one or more of such oligonucleotide probes, and methods of detecting 2019-nCoV and/or diagnosing COVID-19 using the oligonucleotide probes and kits described herein.