Method and System for Improving Specificity of Analyte Detection Using Real-Time Nucleic Acid Amplification
AssigneeGen-Probe Incorporated
InventorsVincent Chabot, David Opalsky
Life of this patent
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Abstract
Methods and systems including programmed computers that can be used for improving nucleic acid analyte detection using real-time amplification and monitoring, where closely related nucleic acid sequences can be resolved from each other. In one embodiment, detection of target sequences differing from each other by a single nucleotide were resolved by applying a mathematical transformation. In another embodiment, two target sequences differing from each other by a single nucleotide were detected and resolved from each other using a labeled probe specific for only one of the two target sequences.
Claims38
A method of determining whether a test sample suspected of comprising nucleic acid templates for a nucleic acid amplification reaction includes a target nucleic acid, the method comprising the steps of:
- (a) acquiring or having acquired a real-time run curve data set comprising signal data representing production of an amplification product in the nucleic acid amplification reaction as a function of a reaction progress parameter,
- wherein the nucleic acid amplification reaction uses any of the target nucleic acid and a non-target nucleic acid that may have been present in the test sample as templates to produce the amplification product;
- (b) calculating or having calculated a first derivative of the real-time run curve data set, including magnitude values of the first derivative;
- (c) comparing or having compared the calculated magnitude values of the first derivative with a first threshold value;
- and
- (d) determining or having determined either that
- the test sample includes the target nucleic acid if any calculated magnitude value of the first derivative met or exceeded the first threshold value, or
- the test sample does not include the target nucleic acid if no calculated magnitude value of the first derivative met or exceeded the first threshold value.
A computer programmed with software instructions to determine whether a target nucleic acid is included in a test sample, the software instructions, when executed by the computer, cause the computer to:
- (a) receive a real-time run curve data set comprising signal data that indicates amplification of the target nucleic acid and a non-target nucleic acid in a nucleic acid amplification reaction as a function of a reaction progress parameter;
- (b) calculate a first derivative of the real-time run curve data set or a processed version thereof, including magnitude values of the first derivative;
- (c) compare the calculated magnitude values of the first derivative with a first threshold value;
- and
- (d) determine either that
- the test sample included the target nucleic acid if any calculated magnitude value of the first derivative met or exceeded the first threshold value, or
- the test sample did not include the target nucleic acid if no calculated magnitude value of the first derivative met or exceeded the first threshold value.
A system that determines whether a target nucleic acid is included in a test sample, the system comprising:
- a nucleic acid analyzer configured to amplify the target nucleic acid and a non-target nucleic acid in a nucleic acid amplification reaction,
- wherein the nucleic acid amplification reaction uses any of the target nucleic acid and the non-target nucleic acid that may have been present in the test sample as templates to produce an amplification product, and
- wherein the nucleic acid analyzer monitors synthesis of the amplification product in the nucleic acid amplification reaction as a function of a reaction progress parameter, whereby there is produced a real-time run curve data set comprising signal data as a function of the reaction progress parameter;
- and
- a computer in communication with the nucleic acid analyzer, the computer being programmed with a set of software instructions causing the computer to
- (a) calculate a first derivative of the real-time run curve data set or a processed version thereof, including magnitude values of the first derivative;
- (b) compare the calculated magnitude values of the first derivative with a first threshold value;
- (c) determine either that
- the test sample included the target nucleic acid if any calculated magnitude value of the first derivative met or exceeded the first threshold value, or
- the test sample did not include the target nucleic acid if no calculated magnitude value of the first derivative met or exceeded the first threshold value;
- and
- (d) generate a non-transient record of the result from (c).
Dependencies are inferred from the claim text.