Multiple amplification cycle detection
Inventors
Rasmussen, Randy P. • Crisp, Robert John • Hemmert, Andrew Clinton • Campbell, Elizabeth Barker • Robbins, Thomas Charles • Eyre, David J.
Assignees
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Publication Number
US-11959132-B2
Publication Date
2024-04-16
Expiration Date
Abstract
Methods and devices are provided for simultaneously amplifying a plurality of sample wells for a predetermined amount of amplification, detecting whether amplification has occurred in a first set of the wells, amplifying for an additional amount of amplification and detecting whether amplification has occurred in a second set of the wells. Methods are also provided for analyzing a target nucleic acid sequence using melt curves that were generated in a plurality of amplification cycles.
Core Innovation
The invention provides a method for analyzing a target nucleic acid in a sample using a two-part sample container that includes a first blister and a second sample well. The second sample well includes primers for amplifying the target nucleic acid, and the sample undergoes a first amplification in the first blister before a portion is moved into the second sample well for amplification conditions through a plurality of amplification cycles.
During amplification in the second sample well, a melt curve of the amplified target nucleic acid is generated. The method repeats the amplification and melt-curve generation steps and determines a Cp by identifying the amplification cycle in which a value for the melt curve exceeds a predetermined value. In additional implementations, the second sample well includes two or more different target nucleic acids with primers that enable simultaneously amplifying the two or more target nucleic acids and generating a plurality of melt curves.
For each of the two or more amplified target nucleic acids, a value for the melt curve is determined and a melt Cp is determined by identifying a melt event in which the value for the melt curve exceeds a predetermined value. In additional implementations, the second sample well further includes a control nucleic acid and primers for amplifying the control nucleic acid, and the method determines values for amplified target and amplified control nucleic acids based on the generated melt curve while repeating through additional amplification cycles.
Claims Coverage
The document contains two independent claims. Across these claims, the main inventive features center on a two-part container workflow, multiplex amplification with multiple melt curves and melt Cp values, and control nucleic acid inclusion with melt-curve-based value determination.
Two-stage blister-to-well amplification with melt-curve Cp determination
providing a sample container comprising a first blister and a second sample well with primers, subjecting the sample to a first amplification in the first blister and moving a portion to the second sample well, subjecting the second sample well to amplification conditions through a plurality of amplification cycles, generating a melt curve, repeating amplification and melt-curve generation, and determining a Cp by identifying the amplification cycle in which a value for the melt curve exceeds a predetermined value
Simultaneous multiplex amplification producing plurality of melt curves and melt Cp values
wherein the second sample well contains two or more different target nucleic acids and primers for amplifying the two or more different target nucleic acids, simultaneously amplifying the two or more target nucleic acids in the second sample well through a plurality of amplification cycles, generating a plurality of melt curves of the amplified two or more target nucleic acids, determining a value for each of the plurality of melt curves from each of the amplified two or more target nucleic acids, and determining a melt Cp for each of the two or more target nucleic acids by identifying a melt event in which the value for the melt curve for each of the two or more target nucleic acids exceeds a predetermined value
Target and control nucleic acid amplification in the second well with melt-curve value determination
providing a sample container comprising a first blister and a second sample well with primers for amplifying the target nucleic acid and a control nucleic acid, subjecting the sample to a first amplification in the first blister and moving a portion to the second sample well, subjecting the second sample well to amplification conditions through at least one amplification cycle to amplify the target nucleic acid and the control nucleic acid in the second sample well wherein the amplification conditions include a dsDNA binding dye, generating a melt curve of the amplified target nucleic acid and amplified control nucleic acid, determining a value for the amplified target nucleic acid, and repeating steps (c), (d), and (e)
Claim coverage is centered on generating melt curves during amplification after a blister-to-well transfer, determining Cp or melt Cp by identifying amplification cycles where melt-curve values exceed predetermined values, extending this to simultaneous multiplex amplification that yields multiple melt curves, and incorporating a control nucleic acid amplified with a dsDNA binding dye such that melt-curve-based target value determination is repeated across cycles.
Stated Advantages
Enables identifying a Cp by identifying the amplification cycle in which a melt-curve value exceeds a predetermined value.
Enables simultaneous multiplex amplification and the generation of a plurality of melt curves for two or more different target nucleic acids, with melt Cp values determined for each target nucleic acid by melt events exceeding predetermined values.
Provides melt-curve-based determination of values for amplified target nucleic acid, and in the control-inclusive method, includes an amplified control nucleic acid under dsDNA binding dye amplification conditions.
Supports distinguishing melt events for multiple targets using melt Cp determination for each amplified target nucleic acid.
Documented Applications
Multiplex nucleic-acid detection using a FilmArray BCID-like protocol/workflow with a two-stage amplification workflow and melt-curve-based Cp/melt calling.
Discriminating clinically relevant infection from background contamination, cross-reactivity, and chromosomal integration, including chromosomal integration versus clinically relevant infection for HHV6 (ciHHV6).
Low-load detection using melt analysis with power-adjusted melt (LED intensity).
Relative or corrected/relative quantification using melt-window integration of a negative first derivative and target/control efficiencies with a relative concentration relationship (ET*Cp,t)/(EC*Cp,c).
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