Differentiation of signals for target nucleic acid sequences
Inventors
Chun, Jong Yoon • Lee, Young Jo
Assignees
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Publication Number
US-11859243-B2
Publication Date
2024-01-02
Expiration Date
Abstract
The present invention relates to differentiating signals of interest for target nucleic acid sequences. The present invention permits to obtain an individual signal value (i.e., variable) contained in a total signal detected at detection temperatures by using mathematical equations. The present invention based on equation-solving approach enables to obtain the individual signal value in a systematical manner, thereby providing analysis results in much more accurate and convenient manner.
Core Innovation
The invention provides a signal-differentiation method for a sample containing two or more target nucleic acid sequences where the signals of interest are not differentiable by a single type of detector. The sample is incubated with signal-generating means for each target nucleic acid sequence, and signals are detected at a first detection temperature and a second detection temperature for the two-target case, or at detection temperatures in the number of N for the N-target case.
The detected total signals at each detection temperature are modeled as equations in variables representing signals of interest generated for each target nucleic acid sequence at each detection temperature. For two targets, the method provides two equations comprising four variables, and for N targets it provides equations in the number of N each comprising variables representing the signals of interest generated at each detection temperature for the target nucleic acid sequences.
To enable differentiation, the method further provides additional equations relating the target-specific variables across detection temperatures via reference values, and it can also use a constraint such as a zero-signal condition with a proviso that the corresponding signal-generating means is prepared to generate no signal at the specified detection temperature. Solutions to at least one variable obtained from the equation sets are then used for differentiating at least one signal of interest and assigning it to at least one of the target nucleic acid sequences.
Claims Coverage
The provided claims include three independent claims: one for differentiating signals for two target nucleic acid sequences, and two for differentiating signals for N target nucleic acid sequences, implemented as a method and as instructions to configure a processor/storage medium. Across these independent claims, the inventive core uses temperature-indexed signal equations with target-specific variables, and additional reference-value or zero-signal constraints to solve for variables and assign signals to target nucleic acid sequences.
Two-target signal differentiation using two detection temperatures and solvable equation sets
Differentiating signals of interest for each of two target nucleic acid sequences that are not differentiable by a single type of detector by incubating the sample with first and second signal-generating means for detection of the two targets, detecting signals at a first detection temperature and a second detection temperature, providing two equations comprising variables representing the signals of interest generated at each detection temperature, providing two additional equations selected from reference-value relationships and a zero-signal condition, and obtaining solutions to at least one variable for differentiating at least one signal of interest to be assigned to at least one of the two target nucleic acid sequences.
N-target signal differentiation using an N-target equation system and additional constraints
Differentiating signals of interest for each of N target nucleic acid sequences in a sample, where N is not less than 2 and the signals are not differentiable by a single type of detector, by incubating the sample with signal-generating means in the number of N, detecting signals at detection temperatures in the number of N, providing equations in the number of N each comprising variables representing the signals of interest at each detection temperature, providing additional equations selected from reference-value relationships and a zero-signal condition, and obtaining solutions to at least one variable for differentiating at least one signal of interest to be assigned to at least one of the target nucleic acid sequences.
Processor-instruction storage medium for N-target signal differentiation
A non-transitory computer readable storage medium containing instructions to configure a processor to perform the N-target signal-differentiation method by receiving signals detected at detection temperatures in the number of N, providing equations in the number of N with variables representing the signals of interest at each detection temperature, providing additional equations selected from reference-value relationships and a zero-signal condition, and obtaining solutions to at least one variable for differentiating at least one signal of interest to be assigned to at least one of the target nucleic acid sequences.
Across the independent claims, the coverage focuses on solving temperature-indexed signal equations using target-specific variables and reference-value relationships, and optionally a zero-signal condition, so that at least one signal of interest is differentiable and assigned to one or more target nucleic acid sequences despite the targets being not differentiable by a single type of detector.
Stated Advantages
Differentiating multi-target signals in a single vessel without melting analysis.
Extracting individual target signal values from total detected signals via equation solving.
Flexibility in selecting signal-generating means.
Using a reference-value pattern across temperatures.
Enables differentiation of signals of interest for two or more target nucleic acid sequences in a sample when the signals are not differentiable for each target nucleic acid sequence by a single type of detector.
Provides separate assignment of at least one signal of interest to at least one target nucleic acid sequence by obtaining solutions to variables from the provided equation sets.
Documented Applications
Experimental demonstrations for extracting/differentiating NG/CT using signals measured at different temperatures (two targets).
Experimental demonstrations for extracting/differentiating NG/CT/MG using signals measured at different temperatures (three targets).
Computer-implemented embodiments demonstrated using real-time PCR implementations including TaqMan real-time PCR and PTOCE real-time PCR, including combined TaqMan/PTOCE.
Two-target differentiation using TaqMan real-time PCR with detection at 60°C and 72°C, producing separate amplification-like curves for each target and enabling presence/absence determination without conventional melting analysis.
Two-target differentiation using PTOCE real-time PCR.
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