Assay devices and methods of use thereof

A self-contained reaction vessel, the self-contained reaction vessel comprising:

• 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 chambers 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.