Assay devices and methods of use thereof

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

• a first layer and a second layer defining a multifunction chamber therebetween, the container provided with magnetic particles and reagents for sample preparation, nucleic acid recovery, and a first-stage nucleic acid amplification reaction, the multifunction chamber comprising a chamber for first-stage nucleic acid amplification, wherein the magnetic particles are provided in a chamber that is fluidly connected to the multifunction chamber or are provided in the multifunction chamber, and wherein the reagents are provided in chambers that are fluidly connected to the multifunction chamber;
• and
• a second-stage reaction zone disposed between the first layer and the second layer and fluidly connected to the multifunction chamber, the second-stage reaction zone comprising a plurality of second-stage reaction chambers different from the chamber for first-stage nucleic acid amplification, each second-stage reaction chamber 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 chambers,
• wherein the first layer and the second layer are flexible barrier films having a water vapor transmission rate (WVTR) in a range of about 0.05 g/m2/24 hrs to about 2 g/m2/24 hrs, and
• wherein the magnetic particles and the reagents are provided in one or more fluid-filled reagent blisters in fluid communication with the multifunction chamber, and wherein the fluid-filled reagent blisters and are filled at the time of manufacture of the container.