Automated analysis of real-time nucleic acid amplification
First Claim
1. A method for determining the presence of a nucleic acid in a sample 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 plot wherein the fluorescent values are recorded for each amplification cycle, performing a confidence band analysis on the plot to generate a positive or negative call, and if the call is positive, confirming the positive call by a melting temperature analysis.
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Abstract
A method and device are 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. A positive result may be verified by melting temperature analysis.
13 Citations
13 Claims
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1. A method for determining the presence of a nucleic acid in a sample 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 plot wherein the fluorescent values are recorded for each amplification cycle, performing a confidence band analysis on the plot to generate a positive or negative call, and if the call is positive, confirming the positive call by a melting temperature analysis.
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10. 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 plot wherein the fluorescent values are recorded for each amplification cycle, calculating slopes of segments of the plot using a plurality of the fluorescent values, using the segment slopes of the 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, outputting a positive result if the fluorescence value of a selected amplification cycle is outside the baseline fluorescence region, and confirming the positive result by melting temperature analysis.
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11. A device for determining the presence of a predetermined nucleic acid in a sample comprising
an instrument for temperature cycling to amplify the nucleic acid, a fluorimeter for detecting fluorescence during amplification of the nucleic acid, the fluorescence obtained from a fluorescent entity capable of providing a signal related to the quantity of the nucleic acid, wherein the fluorimeter measures fluorescence intensity of the fluorescent entity at each of a plurality of amplification cycles, and wherein the fluorimeter measures fluorescence intensity of the fluorescent entity to obtain a melting profile of the nucleic acid, and a processor for performing analysis routines, wherein the processor is programmed to generate a plot of the fluorescent values verses amplification cycle, to perform a confidence band analysis on the plot to generate a positive or negative call, and to perform melting temperature analysis to confirm a positive call.
Specification