Self-contained biological analysis
Inventors
Ririe, Kirk M. • Newswander, Michael R. • Rasmussen, Randy P. • Poritz, Mark A. • Smith, Stewart Benjamin • Kessler, Gary C.
Assignees
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Publication Number
US-11707741-B2
Publication Date
2023-07-25
Expiration Date
Abstract
Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analyses include nucleic acid amplification and detection and immuno-PCR.
Core Innovation
The invention provides a self-contained system for nucleic acid extraction and multiplex PCR, in which one or more sealable ports provide the only access from an exterior so that, when the ports are closed, the system is a fully closed, sealed environment that prevents access from the self-contained system to surrounding atmosphere. The self-contained system includes a cell lysis zone configured for lysing cells or spores in a sample and a nucleic acid preparation zone configured for purifying a plurality of nucleic acids that may be in the sample, with fluid connection from the injector port through the cell lysis zone.
The self-contained system further includes at least one amplification zone configured for amplification of the plurality of nucleic acids that may be in the sample. The sample is introduced via the injector port, the ports are closed after introducing the sample, and the nucleic acids are moved into the amplification zone to mix with a plurality of primer pairs, thermal cycle in the presence of PCR reaction components to create an amplification mixture, and detect which nucleic acids are present in the amplification mixture.
The disclosed embodiments include multiplex nucleic-acid extraction and nested PCR using sealed first-stage and second-stage zones, with nucleic-acid purification using magnetic beads and amplification and detection performed after moving material between zones, and optional adaptation of the system for immuno-PCR (iPCR) by performing antigen capture and washing with DNA-conjugated reporter antibodies inside the sealed pouch.
Claims Coverage
One independent claim is identified. It covers a method that combines nucleic acid extraction with multiplex PCR in a fully closed self-contained sealed system, using sealable ports, a cell lysis zone, a nucleic acid preparation zone, and an amplification/detection zone, with port closure after sample introduction.
Fully closed self-contained sealed system with sealable ports
one or more sealable ports provide the only access from an exterior of the self-contained system such that when all of the one or more ports are closed, the self-contained system is a fully closed, sealed environment that prevents access from the self-contained system to surrounding atmosphere
Injector port fluidly connected through cell lysis to nucleic acid preparation
an injector port for introducing a sample into the self-contained system, and a nucleic acid preparation zone fluidly connected to the injector port through the cell lysis zone
Bead-milling cell lysis zone with rotating blades or paddles
a cell lysis zone configured for lysing cells or spores located in the sample, wherein cells or spores are lysed in the cell lysis zone by impacting the cell lysis zone with rotating blades or paddles for bead-milling
Nucleic acid preparation zone purifying multiple nucleic acids
a nucleic acid preparation zone configured for purifying a plurality of nucleic acids that may be in the sample subsequent to lysis
Multiplex primer mixing in amplification zone
moving the plurality of nucleic acids that may be in the sample into the amplification zone to mix with a plurality of primer pairs in the amplification zone
Thermal cycling with PCR components and primer pairs
thermal cycling the plurality of nucleic acids that may be in the sample in the amplification zone in the presence of PCR reaction components and the plurality of primer pairs to create an amplification mixture
Detection of which nucleic acids are present in the amplification mixture
detecting which of the plurality of nucleic acids are present in the amplification mixture in the amplification zone
Closing ports after sample introduction
closing the one or more ports after introducing the sample
The independent claim’s coverage centers on performing nucleic acid extraction and multiplex PCR inside a fully closed, sealed self-contained environment using sealable ports and a sequence of zone-based operations, with the ports closed after introducing the sample.
Stated Advantages
closing the one or more ports after introducing the sample, supporting a contamination-reducing workflow within a closed environment
Documented Applications
multiplex nucleic-acid extraction and nested PCR
immuno-PCR (iPCR) by performing antigen capture and washing with DNA-conjugated reporter antibodies inside the sealed pouch
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