Self-contained biological analysis
Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analyzes include nucleic acid amplification and detection and immuno-PCR.
1. A container for performing two-stage nucleic acid amplification on a sample in a closed system comprising
one or more sealable ports, the sealable ports providing the only access from an exterior of the container such that when all of the one or more sealable ports is sealed, the container is fully closed, the one or more sealable ports including a sample injection port for introducing the sample into the container,
a cell lysis zone configured for lysing cells or spores located in the sample, the cell lysis zone fluidly connected to the sample injection port,
a nucleic acid preparation zone fluidly connected to the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids,
a first-stage reaction zone fluidly connected to the nucleic acid preparation zone, the first-stage reaction zone comprising a first-stage reaction blister configured for first-stage amplification of the sample, and
a second-stage reaction zone fluidly connected to the first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, 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.
2. The container of claim 1 wherein the first-stage amplification is multiplex amplification for a plurality targets, and each pair of primers in the second-stage reaction chambers is configured for further amplification of one of the first-stage targets.
3. The container of claim 2 wherein each pair of primers in the second stage reaction chamber is configured to be nested within primers used in amplifying the first-stage targets.
4. The container of claim 1 wherein the one or more sealable ports further comprise a second port configured to receive water or reagents, and the sealable ports are configured to be sealed subsequent to introduction of the sample and water or reagents and remain sealed through first- and second-stage amplification.
5. The container of claim 1 wherein the cell lysis zone comprises a cell lysis blister containing a plurality of lysing particles.
6. The container of claim 5 wherein the particles are zirconium silicate beads.
7. The container of claim 6 wherein the nucleic acid preparation zone comprises DNA-binding magnetic beads.
8. The container of claim 1 wherein the blister comprises a flexible material, such that pressure provided on the blister collapses the blister, forcing the contents from the blister.
9. The container of claim 8 wherein the flexible material is a flexible plastic film.
10. The container of claim 1 wherein the second-stage reaction zone further comprises one or more additional second-stage reaction chambers, each of the additional second-stage reaction chambers configured as amplification controls.
11. The container of claim 1 wherein the sealable ports are heat sealable.
12. A container for performing two-stage nucleic acid amplification on a sample in a closed system comprising
one or more sealable ports, the sealable ports providing the only access from an exterior of the container such that when all of the one or more sealable ports is sealed, the container is fully closed, the one or more sealable ports including a sample injection port for introducing the sample into the container,
a first-stage reaction zone fluidly connected to the sample injection port, the first-stage reaction zone comprising a first-stage reaction blister configured for first-stage amplification of the sample, and
a second-stage reaction zone fluidly connected to the first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, 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.
13. The container of claim 12 further comprising a cell lysis zone configured for lysing cells or spores located in the sample, a nucleic acid preparation zone fluidly connected to the cell lysis zone, the nucleic acid preparation zone configured for purifying nucleic acids, the cell lysis zone and the nucleic acid preparation zone both fluidly located between the sample injection port and the first-stage reaction zone.