Detection using concurrent melting curves
Inventors
Galvin, Benjamin William • Robbins, Thomas Charles • Cox, Charles Benjamin
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11398296-B2
Publication Date
2022-07-26
Expiration Date
Abstract
Methods, sample vessels, and instruments are provided for melting curve analysis. A sample is amplified by thermal cycling the sample well between at least an annealing temperature and a denaturation temperature through n cycles, where each cycle includes an in-cycle denaturation step. Fluorescent data is collected during the in-cycle denaturation step of n cycles where n is at least two. Then a composite melting curve is generated using the fluorescent data collected during the denaturation step of each of the n cycles, and the sample is called using the composite melting curve, where the call is a positive or a negative call.
Core Innovation
The invention provides a nucleic-acid testing system for calling a sample positive or negative for a target nucleic acid sequence. A sample well houses a sample including the target nucleic acid and components for amplification, and one or more temperature controlling devices thermally cycle the sample through a plurality of cycles including an in-cycle temperature adjusting segment. During thermal cycling, an optical system detects fluorescence emitted by the sample.
A controller receives data indicative of fluorescence emitted by the sample from the optical system during the in-cycle temperature adjusting segment for two or more cycles. The controller generates a composite melting curve by combining the fluorescence data from the cycles and fits the composite melting curve using an aggregation method. The controller then analyzes the composite melting curve to call the sample negative or positive and displays an indication of the negative or positive call for the sample.
The approach supports calling logic based on features extracted from the composite melting curve, including melt peak analysis and residuals-based evaluation. In some embodiments, the controller calls the sample positive when a melt peak is within a predefined melt window and negative otherwise. In other embodiments, the controller calls the sample positive based on residuals being less than a predefined threshold, including the use of a range-based threshold with an indeterminate call when residuals fall within that range.
Claims Coverage
The patent includes two independent claims, one system claim and one computing-device claim, with the same core sequence: thermal cycling with an in-cycle temperature adjusting segment, fluorescence detection during that segment for two or more cycles, composite melting-curve generation by combining cycle data and fitting using an aggregation method, analysis to call negative or positive, and display of the call.
Composite melting curve from in-cycle fluorescence over multiple cycles
A controller configured to receive data indicative of the amount of fluorescence emitted by the sample from an optical system during the in-cycle temperature adjusting segment for two or more of the plurality of cycles, generate a composite melting curve by combining the data from each of the two or more cycles including fitting the composite melting curve using an aggregation method, analyze the composite melting curve to call the sample negative or positive, and display an indication of the negative or positive call for the sample.
In-cycle temperature adjusting segment with fluorescence detection
One or more temperature controlling devices configured to amplify the sample by thermal cycling the sample well including heating the sample well to a first temperature and cooling the sample well to a second temperature through a plurality of cycles, wherein each cycle includes an in-cycle temperature adjusting segment; and an optical system configured to detect an amount of fluorescence emitted by the sample.
Fitting composite melting curve using an aggregation method
Generate a composite melting curve by combining the data from each of the two or more cycles including fitting the composite melting curve using an aggregation method.
Calling based on melt peak within a predefined melt window
Analyze the composite melting curve to call the sample negative or positive, wherein the controller generates a melt peak and classifies the sample as positive if the melt peak is within a predefined melt window and negative otherwise.
Calling based on residuals threshold
Analyze the composite melting curve and classify the sample as positive when residuals are less than a predefined threshold.
Indeterminate call when residuals fall within a threshold range
The system of claim 6, wherein the controller displays an indeterminate call when the residuals fall within that range.
Computing device instructions for thermocycling and composite melting curve calling
A computing device for calling a sample for a target nucleic acid sequence comprising one or more processors and a non-transitory computer-readable memory storing instructions that cause the computing device to provide control signals to a thermocycling element including heating the sample to a first temperature and cooling the sample to a second temperature through a plurality of cycles with each cycle including an in-cycle temperature adjusting segment; receive data indicative of the amount of fluorescence emitted by the sample during the in-cycle temperature adjusting segment for two or more cycles; generate a composite melting curve by combining the data from each of the two or more cycles including fitting the composite melting curve using an aggregation method; analyze the composite melting curve to call the sample negative or positive; and display an indication of the call for the sample, wherein the call is selected from at least the positive or negative call.
Denaturation/annealing temperature roles in in-cycle segment
Provide control signals to a thermocycling element that include heating the sample to a first temperature and cooling the sample to a second temperature through a plurality of cycles, wherein each cycle includes an in-cycle temperature adjusting segment, wherein the first temperature is a denaturation temperature and the second temperature is an annealing temperature.
Across the independent claims, the inventive concept is centered on calling a sample positive or negative using a composite melting curve constructed by combining fluorescence data collected during an in-cycle temperature adjusting segment for two or more cycles, with fitting using an aggregation method, and applying composite-curve analysis such as melt-window melt peak criteria and residuals-based, including range-based, decision logic with optional indeterminate signaling.
Stated Advantages
Allows calling the sample negative or positive based on analysis of a composite melting curve generated from fluorescence detected during an in-cycle temperature adjusting segment for two or more cycles.
Supports positive/negative classification based on melt peak being within a predefined melt window.
Supports positive classification using residuals being less than a predefined threshold.
Supports an indeterminate call when residuals fall within a threshold range.
Provides an at-least positive/negative call displayed as an indication of the call for the sample.
Documented Applications
FilmArray-like systems and optical systems/thermocycling element configurations are discussed in the provided description as illustrative implementation contexts for detecting fluorescence and generating concurrent/in-cycle melting curves.
Nucleic-acid testing for calling a sample positive or negative for a target nucleic acid sequence using composite melting-curve analysis is explicitly described.
Interested in licensing this patent?