Assay devices and methods of use thereof

A method for amplifying nucleic acids in a sample, comprising:

• providing a container comprising a multifunction chamber and a first reaction zone fluidly connected to the multifunction chamber, the container comprising therein magnetic particles and reagents for sample preparation, nucleic acid recovery, and a first-stage nucleic acid amplification reaction, wherein the magnetic particles and the reagents are fluidly connected to the multifunction chamber or the first reaction zone or both;
• introducing the sample into the multifunction chamber;
• and
• performing at least two steps in the multifunction chamber, wherein the steps include:
• (1) contacting the sample and the magnetic beads prior to lysis,
• (2) generating a lysate in the presence of the magnetic particles,
• (3) binding nucleic acids with the magnetic particles,
• (4) isolating the magnetic particles from the lysate,
• (5) performing at least one wash on the magnetic particles isolated from the lysate, and
• (6) amplifying nucleic acids in a first-stage nucleic acid amplification reaction,
• wherein the multifunction chamber is provided with lysis particles.

A system for performing nucleic acid amplification on a sample, the system comprising:

• a reaction vessel that includes:
• a sample lysis zone configured for lysis of cells or spores present in a sample,
• a first reaction zone fluidly connected to the sample lysis zone, the first reaction zone configured for recovering nucleic acids from a lysed sample and for first-stage amplification of nucleic acids present in the first reaction zone,
• a second-stage reaction zone fluidly connected to the first reaction zone but separate from the first reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction wells, each second-stage reaction well comprising a pair of primers configured for further amplification of the sample, the second stage reaction zone configured for contemporaneous thermal cycling of all of the plurality of second-stage reaction wells, and
• a plurality of liquid reagent blisters fluidly connected to one or more of the sample lysis zone, the first reaction zone, or the second-stage reaction zone, wherein liquid reagents are provided in the liquid reagent blisters at time of manufacture,
• wherein the self-contained reaction vessel comprises a first flexible barrier film layer at least partially bonded to a second flexible barrier film layer with the sample lysis zone, first reaction zone, second-stage reaction zone, and the plurality of liquid reagent blisters comprising open spaces between the first and second flexible barrier film layers, wherein the first and second flexible barrier film layers have a water vapor transmission rate (WVTR) in a range of about 0.05 g/m2/24 hrs to about 2 g/m2/24 hrs,
• a thermocycling instrument that includes:
• a receptacle for positioning the reaction vessel in the instrument;
• a heater/cooler positionable in the instrument for heating and/or cooling one or more of the sample lysis zone, the first reaction zone, or the second-stage reaction zone,
• a cell lysis component configured for generating a lysate in the reaction vessel, and
• a fluid movement component configured for moving fluids in the flexible container between at least the one or more reagent blisters, the sample lysis zone, the first reaction zone, or the second-stage reaction zone.