High density self-contained biological analysis
Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analyses include high density nucleic acid amplification and detection and immuno-PCR.
1. A container for performing a plurality of reactions on a fluid sample in a closed system comprising
a high density reaction zone provided in the closed system comprising
a channel for receiving the sample into the high density reaction zone,
a plurality of high density reaction wells fluidly connected to the channel, each high density reaction well comprising one or more reagents, and
a barrier layer disposed between the channel nd the high density reaction wells that minimizes cross-contamination between high density reaction wells upon introduction of the sample to the high density reaction zone, wherein the barrier layer is a pierced layer having one or more piercings per reaction well, and wherein the piercings extend through the pierced layer and are large enough to allow the fluid sample to pass in the presence of a force, but small enough to substantially prevent the fluid sample from passing absent the force,
wherein the high density reaction zone is configured such that the sample received through the channel flows across the pierced layer, over all of the piercings, and through the piercings into the high density reaction wells.
2. The container of claim 1 wherein the container comprises a flexible layer that can be pressed against the high density reaction wells to seal a portion of the sample in each of the plurality of wells.
3. The container of claim 1 wherein the force is vacuum in each of the high density reaction wells.
4. The container of claim 1 wherein the plurality of high density reaction wells comprises at least 50 high density reaction wells.
5. The container of claim 1 wherein the plurality of high density reaction wells comprises at least 120 high density reaction wells.
6. The container of claim 1 wherein the plurality of high density reaction wells comprises at least 400 high density reaction wells.
7. The container of claim 1 wherein the reagents comprise a pair of PCR primers.
8. The container of claim 7 wherein one well contains a control nucleic acid and a pair of primers configured to amplify the control nucleic acid, and at least one other well contains the same primers but does not contain the control nucleic acid.
9. The container of claim 8 wherein the control nucleic acid is a synthetic nucleic acid.
10. The container of claim 1 wherein each of a plurality set of the high density reaction wells contains a different pair of PCR primers.
11. The container of claim 10 further comprising
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, and
a first-stage reaction zone fluidly connected to the nucleic acid preparation zone and the high density reaction zone, the first-stage reaction zone comprising a first-stage reaction blister configured for first-stage amplification of the sample.
12. The container of claim 1 wherein the plurality of high density reaction wells are formed in a high density array, each of the wells in the high density array being sealed on a first end of the high density array and the pierced layer is on a second opposite end of the high density array.
13. The container of claim 12 wherein the high density array is formed from a piece of plastic and the plurality of wells are formed in the piece of plastic.
14. The container of claim 11 wherein the injection port is sealable to provide for the closed system.
15. The container of claim 14 wherein the cell lysis zone, the nucleic acid preparation zone, the first stage reaction zone, and the high density reaction zone are all provided within the closed system.
16. The container of claim 1 , further comprising a sample injection port of introducing the sample into the container, wherein the injection port is sealable, thereby creating the closed system when sealed.
17. The container of claim 1 , wherein the piercings are 0.001-0.1 mm across.
18. The container of claim 17 , wherein the piercings are 0.005-0.02 mm across.
19. The container of claim 1 , wherein the pierced layer includes only one piercing per reaction well.