Methods and apparatus for sequential amplification reactions
Inventors
Smith, Joseph H. • Persing, David H. • Wortman, Alan • Chang, Ronald • Swenson, David
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Assignees
Member
CepheidCepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Cepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Publication Number
US-12123052-B2
Publication Date
2024-10-22
Expiration Date
Abstract
The invention provides methods and apparatus for carrying out multiple amplification reactions in a single reaction chamber by successive cycles of loading reaction mixture, amplifying, and removing spent reaction mixture in a fluidly closed reaction system. In particular, the present invention allows amplification of a plurality of target polynucleotides from a single sample by carrying out under closed-loop control successive amplifications of different target polynucleotides from different portions of the sample.
Core Innovation
The invention provides methods and apparatus for carrying out multiple amplification reactions in a single reaction chamber by successive cycles of loading reaction mixture, amplifying, and removing spent reaction mixture in a fluidly closed reaction system. The present invention allows amplification of a plurality of target polynucleotides from a single sample by carrying out successive amplifications of different target polynucleotides from different portions of the sample.
The patent addresses problems with existing approaches to amplifying multiple targets from a single sample, including the difficulty of finding reaction conditions suitable for simultaneous amplification (multiplex PCR), constraints on mid-course adjustments to favor different reactants, the manufacturing complexity and fluidic valving of microfluidic aliquoting approaches, and the increased contamination risk when reactions are accessed after initiation or when multiple ports are used. It further emphasizes the importance of fluidly closed reaction conditions to minimize false positive assessments in applications such as intraoperative testing and infectious agent testing.
Key elements disclosed include automated control of sequential amplification reactions using an indicator that generates an optical signal related to amplicon quantity, monitoring that optical signal, and automatically removing and replacing reaction mixtures when the optical signal reaches a predetermined level. The invention further contemplates a fluidly closed reaction system employing reservoirs, a pump and rotary valve under microprocessor control, and computer-readable programs to implement closed-loop control, and includes measures to reduce foaming and bubble formation between successive reactions such as purging and heating of the empty reaction chamber ([procedural detail omitted for safety]).
Claims Coverage
Independent claims identified in the patent: 1, 7, 16, and 20. Four main inventive features were extracted from these independent claims.
Automated optical‑signal‑triggered sequential amplification in a single reaction chamber
Providing a reaction chamber suitable for optical interrogation that is selectably in fluid communication with a waste reservoir, a sample reservoir, and a plurality of reagent reservoirs; loading a reaction mixture by mixing a sample portion and amplification reagents for a target polynucleotide; amplifying the target polynucleotide in the presence of an indicator capable of generating an optical signal related to amplicon quantity; monitoring the optical signal; and automatically removing the reaction mixture to the waste reservoir and loading the chamber with a subsequent reaction mixture whenever the optical signal reaches or exceeds a predetermined level, repeating these steps until the plurality of amplification reactions has been carried out.
Fluidly closed sequential amplification for detecting multiple target polynucleotides
Providing a reaction chamber in a fluidly closed reaction system; mixing a portion of the sample with amplification reagents to form a reaction mixture; amplifying one or more target polynucleotides in the reaction chamber to form amplicons; detecting the amplicons to determine presence or absence of the target polynucleotides; and repeating mixing, amplifying, and detecting steps until the presence or absence of the plurality of target polynucleotides is determined.
Sequential aliquot‑based amplification from a single sample in a fluidly closed system
Providing a reaction chamber selectably in fluid communication with a waste reservoir, a sample reservoir containing a sample, a first reactant reservoir containing first amplification reagents, and a second reactant reservoir containing second amplification reagents, each reservoir being fluidly closed; combining a first portion of the sample with first amplification reagents to form a first reaction mixture and subjecting it to amplification; fluidly transferring the first reaction mixture to the waste reservoir; combining a second portion of the sample with second amplification reagents to form a second reaction mixture and subjecting it to amplification; wherein detection of the first and second amplicons determines presence or absence of the plurality of target polynucleotides.
Sequential organism identification with automatic continuation or termination based on optical signal
Amplifying one or more target polynucleotides from a portion of a sample in the presence of an indicator that generates an optical signal related to amplicon quantity; monitoring the optical signal; automatically removing the reaction mixture and transferring a subsequent reaction mixture when the optical signal reaches or exceeds a predetermined level, otherwise automatically terminating the sequence of amplification reactions; and repeating until the sequence has been carried out or terminated.
The independent claims collectively cover performing multiple, sequential amplification reactions in a fluidly closed reaction system using optical‑signal monitoring of indicators to trigger automated removal and reloading of reaction mixtures, conducting sequential aliquot‑based analyses from a single sample, and enabling automatic continuation or termination of reaction sequences based on monitored optical signals.
Stated Advantages
Enables amplification of a plurality of target polynucleotides from a single sample by successive amplifications of different targets in a single reaction chamber.
Maintains fluidly closed reaction conditions to minimize contamination and reduce false positive assessments.
Provides automated closed‑loop control based on real‑time optical indicators to trigger termination and initiation of successive reactions, allowing rapid negative or conditional test decisions.
Reduces or eliminates foam and bubble formation in the reaction chamber between successive reactions by purging and heating the empty reaction chamber ([procedural detail omitted for safety]).
Documented Applications
Clinical and biological research applications including detection and monitoring of infectious diseases.
Detection of mutations and detection of cancer markers, including detection of minimal residual disease in cancer patients.
Environmental monitoring and genetic identification.
Detection of pathogens in biodefense applications, including infectious agent testing and rapid detection of bio‑terror threats.
Food pathogen testing and blood supply screening.
Intraoperative sample testing where fluidly closed reaction conditions are important to minimize false positives.
Exemplary use case: detection of methicillin‑resistant Staphylococcus aureus (MRSA) by successive amplification reactions in the same reaction chamber (examples and instrumentation results provided).
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