Method and apparatus for detecting a plurality of target nucleic acid sequences in sample
Inventors
KO, Sung Moon • Han, Ji Hye • Kim, Young Wook • PARK, Young Yong
Assignees
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Publication Number
US-12188082-B2
Publication Date
2025-01-07
Expiration Date
Abstract
The method of the present invention enables efficient detection of a plurality of target nucleic acid sequences in one detection channel, by obtaining a data set of cycle/signal-change value.
Core Innovation
The invention provides a method for detecting a plurality of target nucleic acid sequences in a sample by contacting the sample with signal-generating compositions in a single reaction vessel. The signal-generating compositions form a plurality of duplexes for the plurality of target nucleic acid sequences, where each duplex provides a signal indicating the presence or absence of its corresponding target and the duplexes have different Tm values. A real-time amplification reaction is performed for at least two cycles to support both formation of the duplexes and a hybridization and/or dissociation of the formed duplexes.
The method obtains signal values at all or partial cycles across at least two detection temperatures for each of a plurality of detection temperature ranges assigned to the plurality of target nucleic acid sequences. Each detection temperature range includes a temperature range in which the amount of a duplex for the corresponding target nucleic acid sequence changes. From the signal values, a signal-change value is obtained between two reference temperatures selected from the at-least-two detection temperatures in each detection temperature range, thereby producing a data set of cycle/signal-change value for each target.
Using the data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences, the method determines the presence or absence of the target nucleic acid sequences in the sample. The disclosed approach also includes embodiments that refine detection and analysis by using duplex Tm differences with assigned detection temperature ranges, selecting reference temperatures from multiple detected temperatures, and computing the signal-change value to support presence/absence determination, including options noted for restoring cycle/signal datasets and combining duplex-based signaling for some targets with cleavage-based signaling for others.
Claims Coverage
Independent claim clm-00001 defines the overall inventive concept with five main inventive elements: single-vessel duplex formation with different Tm values, real-time amplification for at least two cycles including duplex hybridization/dissociation, multi-temperature detection across detection temperature ranges assigned to targets, computation of a cycle/signal-change dataset using reference temperatures, and determining presence/absence using that dataset. The remaining dependent claims further specify mechanisms for duplex signaling, temperature/reference selection constraints, and the way signal-change value is computed.
Single-vessel target duplex formation with different Tm values
Contacting in a single reaction vessel the sample to signal-generating compositions for detecting the plurality of target nucleic acid sequences; wherein the signal-generating compositions form a plurality of duplexes for the plurality of target nucleic acid sequences, each duplex provides a signal indicating the presence or absence of each corresponding target nucleic acid sequence and the plurality of duplexes have Tm values different from each other.
Real-time amplification with duplex formation and hybridization/dissociation
Performing a real-time amplification reaction for at least two cycles for (i) the formation of the plurality of duplexes and (ii) a hybridization and/or dissociation of the formed duplexes.
Multi-detection temperatures in target-assigned detection temperature ranges
Obtaining at all or partial cycles of the reaction, signal values at at least two detection temperatures in each of a plurality of detection temperature ranges assigned to the plurality of target nucleic acid sequences; wherein each detection temperature range comprises a temperature range in which the amount of a duplex for each corresponding target nucleic acid sequence is changed.
Signal-change value between reference temperatures to generate cycle/signal-change datasets
Obtaining a signal-change value of signal values between two reference temperatures selected from the at least two detection temperatures in each detection temperature range such that a data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences is obtained.
Presence/absence determination from cycle/signal-change datasets
Determining the presence or absence of the plurality of target nucleic acid sequences in the sample by the data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences.
The claim set centers on using multiple target-specific duplexes having different Tm values in a single reaction vessel, obtaining signals across target-assigned detection temperature ranges during real-time amplification, converting multi-temperature signals into a cycle/signal-change dataset via signal-change values between reference temperatures, and using that dataset to determine presence or absence for each target. Dependent claims refine how duplex signaling is generated, how reference temperatures are selected, and how the signal-change value is calculated.
Stated Advantages
Reduction of false positives is claimed in the provided document summary/experimental examples.
Documented Applications
Nucleic-acid detection assays for multiple targets using kits and Ct determination are described in the provided document summary/experimental examples, including multi-target detection and evaluation of accuracy/false-positive reduction.
Detection of specific target nucleic acid sequences including Chlamydia trachomatis (CT), Treponema pallidum (TP), HBB, and HPV types 35/18/33/39/52 is described in the provided document summary/experimental examples.
Device and computer-readable storage medium implementations are described as part of the disclosed embodiments in the provided document summary.
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