Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12522866-B2
Publication Date
2026-01-13
Expiration Date
Abstract
Provided herein are methods and compositions to determine the efficacy of a nucleic acid pre-amplification reaction.
Core Innovation
The disclosure relates to methods, compositions, and kits for verifying efficacy of a nucleic-acid pre-amplification reaction by using a PreAmp Spike-In Control (PASIC). A first nucleic acid template is intended to be pre-amplified, while a second nucleic acid template is included in the first amplification mixture but is not amplified during the first amplification reaction. After the first amplification reaction, the product mixture is used for a quantitative second nucleic acid assay to evaluate the relative amounts of the unamplified control template and the amplification product derived from the first template.
A second quantitative nucleic acid assay is performed using reagents for a quantitative assay, including qPCR/quantitative PCR and quantitative hybridization using labeled probes. The signal from the second assay is used to generate an amount indicative of the second nucleic acid template, and a signal indicative of the amount of the amplification product of the first nucleic acid template is also determined in the same second mixture. A condition in the first amplification reaction is detected based on comparing the difference between these measured amounts, such that the condition is detected when the difference is less than a threshold value.
The disclosed PASIC configurations include separate versus overlapping templates on a single nucleic acid molecule, multiplex operation, and considerations relating to template length/content. The pre-amplification step is described for either PCR or reverse transcription, and the quantitative comparison can be expressed using cycle- or amount-based differences such as ΔCq. Example data are provided in inhibitor-containing samples, including blood and chocolate extract, with TBP amplification used to demonstrate detection of inhibition.
Claims Coverage
The independent claim describes a two-stage, quantitative second-assay detection scheme using a first amplification reaction that amplifies a first nucleic acid template while leaving a second nucleic acid template unamplified, followed by quantitative measurement of both in a second nucleic acid assay and detection based on whether their measured difference is less than a threshold value. This claim includes three inventive features centered on the unamplified spike-in control, the quantitative second assay, and the threshold-based comparison.
Unamplified second nucleic acid template included in the first amplification reaction
The method assembles a first mixture comprising a first nucleic acid template, a second nucleic acid template, and a first set of primers specific for the first nucleic acid template, and carries out the first amplification reaction for a first number of cycles to generate a product mixture comprising the second nucleic acid template and an amplification product of the first nucleic acid template, wherein the second nucleic acid template is not amplified in the first amplification reaction.
Quantitative second nucleic acid assay measuring the unamplified template amount
The method assembles a second mixture comprising at least a portion of the product mixture and reagents for a quantitative second nucleic acid assay, carries out the second nucleic acid assay to generate a signal indicative of the amount of the second nucleic acid template in the second mixture, and determines a signal indicative of the amount of the amplification product of the first nucleic acid template in the second mixture.
Threshold-based detection from the difference between measured template and amplification product amounts
The method detects a condition in the first amplification reaction when the difference between the amount of the amplification product of the first nucleic acid template and the amount of the second nucleic acid template in the second mixture is less than a threshold value.
Across the independent claim, the core coverage centers on including an unamplified second nucleic acid template as a spike-in control during a first amplification reaction, quantifying both the unamplified template and the amplification product in a quantitative second nucleic acid assay, and detecting a condition using a threshold comparison of the measured difference.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?