Methods for quantitative amplification
Inventors
Spaulding, Usha K. • Rogatcheva, Margarita • Poritz, Mark Aaron • Crisp, Robert John
Assignees
Biomerieux SA • Biofire Defence LLC • Biofire Defense LLC • Biofire Diagnostics LLC
Publication Number
US-11268141-B2
Publication Date
2022-03-08
Expiration Date
2037-03-20
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Abstract
Methods, sample vessels, and instruments are provided for quantitative and semi-quantitative amplification.
Core Innovation
The invention provides methods, sample vessels, and instruments for quantitative and semi-quantitative amplification of nucleic acids. Specifically, it introduces a workflow in which internal quantification standard nucleic acids—each at known, distinct concentrations—are included within a multiplex amplification mixture along with target primers. The first-stage mixture is then divided into second-stage reactions to separately amplify both targets and quantification standards, enabling generation of a standard curve within the same overall reaction.
The problem addressed is the difficulty of quantitatively detecting and distinguishing pathogenic from commensal loads in clinical samples using traditional or standard PCR-based diagnostics. Previous multiplex PCR approaches faced challenges in robust quantification, particularly due to the inability to include multiple, physically separated standard curve reactions within integrated systems like FilmArray, where only a single primary reaction chamber is available. External standard curves that require separate dilution chambers are not feasible in such closed, single-reaction platforms.
By embedding multiple internal quantification standards directly into the multiplexed reaction, the described methods generate an internal standard curve that can account for both assay-specific and matrix-derived variabilities. The innovation also allows sample processing controls—such as natural or synthetic microorganisms added in known amounts—to serve as quantification standards, ensuring that extraction efficiency and matrix inhibition are included in quantitative calculations. The invention extends to specialized sample vessels and automated instruments configured for these integrated processes.
Claims Coverage
The patent claims focus on multiple inventive features for quantitative and semi-quantitative nucleic acid amplification, as reflected in the independent claims.
Quantitative two-step amplification using internal quantification standard nucleic acids
A method wherein a first-stage multiplex amplification mixture contains target primers and a plurality of internal quantification standard nucleic acids, each standard present at a distinct known concentration and different sequence, along with at least one quantification standard primer. After amplification, the mixture is divided into second-stage individual reactions—some for targets, some for standards—and amplification is performed to generate both target and standard amplicons. The quantification standard amplicons are used to generate a standard curve for quantification.
Quantitative amplification with generation of standard curve from quantification standard amplicons
A method in which an amplification mixture contains a pair of target primers for a target and a plurality of quantification standard nucleic acids (each with a different sequence and known concentration) and at least one pair of quantification standard primers. Amplification produces target and quantification standard amplicons; a standard curve is generated from the quantification standards, which is used to quantify the target nucleic acid.
These inventive features collectively allow quantitative amplification and internal standard curve generation using distinct quantification standards within multiplex or singleplex reactions, facilitating target quantification without the need for external standard curves.
Stated Advantages
Allows simultaneous quantification of multiple target species in a multiplex reaction by generating an internal standard curve using internal quantification standards.
Accounts for assay-specific and matrix-derived variances in PCR outcomes, providing robust and accurate quantification even in complex or inhibitory sample matrices.
Integrates sample processing controls as quantification standards, enabling quantification that includes extraction efficiency and workflow losses.
Eliminates the need for setting up multiple external standard curves or physically separated dilution reactions, facilitating operation in systems with a single primary amplification chamber.
Provides quantitative and semi-quantitative outputs (such as specific concentrations or standardized bins) in integrated, sample-to-answer systems.
Documented Applications
Quantitative and semi-quantitative detection and measurement of multiple pathogens and targets in clinical specimens using integrated multiplex PCR systems, such as respiratory, blood, gastrointestinal, or meningitis panels.
Use in human, veterinary, industrial, and environmental samples for detection and quantification of nucleic acids from a wide range of organisms.
Application for process optimization and evaluation of sample processing methods by tracking recovery and extraction efficiency using quantification standards through all workflow steps.
Use for back-calculating true titers of analytes in a sample by factoring in losses during nucleic acid extraction, enabling more accurate quantification.
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