Variant classification through high-confidence mutation detection from fluorescence signals measured with a multiple mutation assay
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Publication Number
US-12624402-B2
Publication Date
2026-05-12
Expiration Date
Abstract
A system and method for SARS-CoV-2 variant classification through mutation detection from qPCR fluorescence signals. The system receives fluorescence signals, wherein a first fluorescence signal indicates quantitative presence of a first genomic region as a control for SARS-CoV-2, and a second fluorescence signal indicates quantitative presence of a second genomic region of a first mutation present in a subset of variants of SARS-CoV-2. The system measures a first Cq for the first fluorescence signal and a second Cq for the second fluorescence signal as the signals cross a threshold RFU. The system calculates a delta Cq as a difference between the two. Further, the system identifies a first peak RFU for the first fluorescence signal and a second peak RFU for the second fluorescence signal, and calculates a RFU ratio of the two. The system detects presence of the first mutation based on the delta Cq and/or the RFU ratio.
Core Innovation
The invention relates to a SARS-CoV-2 multiple mutation qPCR variant classification system that uses fluorescence signals measured over a plurality of thermal cycles for a test sample. A first fluorescence signal indicates quantitative presence of a first genomic region as a control for SARS-CoV-2, and a second fluorescence signal indicates quantitative presence of a second genomic region of a first mutation present in a subset of variants of SARS-CoV-2. The system measures quantitative cycle (Cq) values for both fluorescence signals as each signal crosses a threshold relative fluorescence unit (RFU).
From the fluorescence data, the system identifies a first peak RFU for the first fluorescence signal and a second peak RFU for the second fluorescence signal. It calculates a delta Cq as a difference between the second Cq and the first Cq, and calculates a RFU ratio as the second peak RFU to the first peak RFU. Detection of presence of the first mutation is based on the delta Cq being below a Cq tolerance or the RFU ratio being above a RFU tolerance.
The system thereby performs high-confidence mutation detection for a mutation pattern by combining control-normalized Cq behavior (delta Cq) with peak RFU information (RFU ratio). It searches known variants by matching mutation patterns and, if no match is identified, indicates a candidate new variant for genetic sequencing.
Claims Coverage
The partial content includes three independent claim families: a method for detecting a mutation in SARS-CoV-2 using delta Cq and RFU ratio, a non-transitory computer-readable storage medium with corresponding instructions, and a system with corresponding operations. Across the independent claims, the coverage centers on the same core inventive analysis: control-based normalization using delta Cq together with a peak RFU ratio thresholding decision rule for detecting mutation presence in a subset of SARS-CoV-2 variants.
Control and mutation genomic region fluorescence signal reception for qPCR
Receiving fluorescence signals over a plurality of thermal cycles of a quantitative Polymerase Chain Reaction (qPCR) for a test sample, wherein a first fluorescence signal indicates quantitative presence of a first genomic region as a control for SARS-COV-2, and a second fluorescence signal indicates quantitative presence of a second genomic region of a first mutation present in a subset of variants of SARS-COV-2.
Dual Cq measurement using threshold RFU crossing
Measuring a first quantitative cycle (Cq) for the first fluorescence signal as the first fluorescence signal crosses a threshold relative fluorescence unit (RFU) and measuring a second Cq for the second fluorescence signal as the second fluorescence signal crosses the threshold RFU.
Peak RFU identification and ratio computation
Identifying a first peak RFU for the first fluorescence signal and identifying a second peak RFU for the second fluorescence signal; and calculating a RFU ratio of the second peak RFU to the first peak RFU.
Delta Cq calculation and mutation detection by tolerance rule
Calculating a delta Cq as a difference between the second Cq and the first Cq; and detecting presence of the first mutation based on the delta Cq being below a Cq tolerance or the RFU ratio being above a RFU tolerance.
Computer-implemented mutation detection instructions on non-transitory storage
Storing instructions on a non-transitory computer-readable storage medium that, when executed by a computer processor, cause the computer processor to perform operations comprising receiving fluorescence signals, measuring first and second Cq values at threshold RFU crossing, identifying first and second peak RFU values, calculating delta Cq and RFU ratio, and detecting presence of the first mutation based on the delta Cq and/or RFU ratio tolerance rule.
qPCR analysis system performing delta Cq and RFU ratio mutation detection
A system comprising a computer processor and a non-transitory computer-readable storage medium storing instructions that, when executed, cause the computer processor to perform operations comprising receiving fluorescence signals, measuring first and second Cq values at threshold RFU crossing, identifying first and second peak RFU values, calculating delta Cq and RFU ratio, and detecting presence of the first mutation based on the delta Cq and/or RFU ratio tolerance rule.
Across the independent claims, the invention is directed to qPCR-based mutation detection for SARS-CoV-2 using two signals corresponding to a control genomic region and a mutation genomic region, computing delta Cq and an RFU peak ratio, and applying a tolerance-based decision rule.
Stated Advantages
High-confidence mutation detection by combining delta Cq-based control normalization with RFU ratio thresholding.
True positive versus false positive discrimination using delta Cq and/or peak RFU ratio.
Documented Applications
Searching known variants by matching mutation patterns and indicating a candidate new variant for genetic sequencing when no match is found.
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