Back to results
US 6,387,621 B1Granted
Automated analysis of real-time nucleic acid amplification
AssigneeUniversity of Utah Research Foundation
InventorCart T. Wittwer
Life of this patent
Estimating the term…
Abstract
A method is described for analyzing a sample for the presence of a nucleic acid wherein the sample is amplified, illustratively using PCR, in the presence of a fluorescent probe capable of detecting the presence of the nucleic acid sample. A baseline region is determined by comparing the fluorescence at various amplification cycles, and the fluorescence at a selected amplification cycle is compared to the baseline region to determine whether the fluorescence measurement falls outside of that baseline region, indicating the presence of the nucleic acid.
Claims24
A method for determining the presence of a nucleic acid comprising the steps of
- providing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a signal related to the quantity of the nucleic acid,
- amplifying the nucleic acid through a plurality of amplification cycles in the presence of the fluorescent entity,
- measuring fluorescence intensity of the fluorescent entity at each of the plurality of amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle,
- generating a fluorescence-verses-amplification-cycle plot wherein the fluorescent values are recorded for each amplification cycle,
- calculating slopes of segments of the fluorescence-verses-amplification-cycle plot using a plurality of the fluorescent values,
- using the segment slopes of the fluorescence-verses-amplification-cycle plot to establish a baseline fluorescence region by generating a slope value for each of a plurality of the amplification cycles, and establishing the baseline fluorescence region comprising an interval of cycles that includes the amplification cycle with the slope value having an absolute value closest to zero, and
- ascertaining whether the fluorescence value during a selected amplification cycle is outside the baseline fluorescence region.
4 more elements
A method for determining the presence of a nucleic acid comprising the steps of
- providing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a signal related to the quantity of the nucleic acid,
- initially amplifying the nucleic acid through a set number of amplification cycles in the presence of the fluorescent entity,
- measuring fluorescence intensity of the fluorescent entity at each of the amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle,
- generating a fluorescence-verses-amplification-cycle plot wherein the fluorescent values are recorded for each amplification cycle,
- calculating slopes of segments of the fluorescence-verses-amplification-cycle plot using a plurality of the fluorescent values,
- using the slopes to identify amplification cycles for use in establishing a baseline fluorescence region,
- ascertaining whether a positive result is indicated by determining whether the fluorescence value during any of the of amplification cycles is outside the baseline fluorescence region, and
- continuing amplification through additional amplification cycles and repeating the measuring, determining, and ascertaining steps after each additional amplification cycle until either the positive result is obtained or a maximum cycle number is reached.
5 more elements
An automated method for determining the presence of a nucleic acid comprising the steps of
- placing a sample into a container containing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a signal related to the quantity of the nucleic acid,
- placing the container into a device for amplifying the nucleic acid through a plurality of amplification cycles in the presence of the fluorescent entity,
- measuring fluorescence intensity of the fluorescent entity at each of the plurality of amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle,
- generating a fluorescence-verses-amplification-cycle plot wherein the fluorescent values are recorded for each amplification cycle,
- calculating slopes of segments of the fluorescence-verses-amplification-cycle plot using a plurality of the fluorescent values,
- using the segment slopes of the fluorescence-verses-amplification-cycle plot to establish a baseline fluorescence region by generating a slope value for each of a plurality of the amplification cycles, and establishing the baseline fluorescence region comprising an interval of cycles that includes the amplification cycle with the slope value having an absolute value closest to zero, and
- outputting a positive result if the fluorescence value of a selected amplification cycle is outside the baseline fluorescence region.
4 more elements
A method for determining the presence of a nucleic acid comprising the steps of
- providing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a fluorescence intensity related to the quantity of the nucleic acid,
- amplifying the nucleic acid through a plurality of amplification cycles in the presence of the fluorescent entity,
- measuring the fluorescence intensity of the fluorescent entity at each of the plurality of amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle,
- generating an array wherein the fluorescent values are recorded for each amplification cycle,
- calculating first derivative values of a plurality windows of the array using a plurality of the fluorescent values,
- establishing a baseline confidence interval using an interval of windows that includes the window having a first derivative absolute value closest to zero, and
- ascertaining whether the fluorescence value produced during a selected amplification cycle is outside the baseline confidence interval.
4 more elements
A method for determining the presence of a nucleic acid comprising the steps of
- providing a fluorescent entity capable of indicating the presence of the nucleic acid and capable of providing a signal related to the quantity of the nucleic acid,
- amplifying the nucleic acid through a plurality of amplification cycles in the presence of the fluorescent entity,
- measuring fluorescence intensity of the fluorescent entity at each of the plurality of amplification cycles to produce a fluorescent value for each cycle related to the quantity of the nucleic acid present at each cycle,
- generating a well-behaved fluorescence-verses-amplification-cycle plot wherein the fluorescent values are recorded for each amplification cycle,
- determining a cutoff cycle,
- calculating slopes of segments of the fluorescence-verses-amplification-cycle plot using a plurality of the fluorescent values,
- using the slopes of segments to assign slope values for a plurality of the amplification cycles prior to the cutoff cycle,
- choosing the fluorescent intensity measurement of the amplification cycle with the slope value having and absolute value closest to zero for establishing a baseline fluorescence region, and
- ascertaining whether the fluorescence value during a selected amplification cycle is outside the baseline fluorescence region.
6 more elements
Dependencies are inferred from the claim text.