Self-contained biological analysis
View Patent ↗Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analysis include nucleic acid amplification and detection and immuno-PCR.
1. A method of amplifying nucleic acids in a sample, comprising the steps of
a. providing a container having
i. a plurality of fluidly connected reaction blisters including an amplification blister,
ii. an additional set of second-stage amplification chambers fluidly connected to the amplification blister, each additional second-stage amplification chamber configured for further amplifying a specific nucleic acid that may be present in the sample, and
iii. one or more sealable ports fluidly connected to the reaction blisters, the sealable ports providing the only access from an exterior of the container to the reaction blisters such that when all of the one or more sealable ports is sealed, the container is fully closed,
b. injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,
c. extracting the nucleic acids from the sample, and moving the extracted nucleic acids to the amplification blister,
d. subjecting the nucleic acids in the amplification blister to amplification conditions,
e. fluidly moving a portion of the nucleic acids to each of the additional second-stage amplification chambers,
f. performing second-stage amplification in the additional second-stage amplification chambers, and
wherein the extracting step includes lysing cells contained within the sample to generate a lysate,
mixing nucleic acid-binding magnetic beads with the lysate and incubating to allow the nucleic acids to bind to the magnetic beads,
deploying a magnet adjacent to one of the plurality of blisters to contain the magnetic beads in that blister, and
moving the remainder of the lysate to another of the plurality of blisters.
2. The method of claim 1 wherein the second-stage amplification chambers each comprise a pair of primers, and the amplifying subjecting the nucleic acids step and second-stage amplification include thermal cycling for PCR amplification.
3. The method of claim 2 wherein the amplifying subjecting the nucleic acids step is terminated prior to reaching log phase, and the second-stage amplification proceeds to log phase.
4. The method of claim 1 wherein the lysing step includes repeatedly impacting an exterior surface of the container by way of a paddle to generate high velocity impacts.
5. The method of claim 1 wherein the subjecting the nucleic acids step further comprises detecting amplification of nucleic acids in the sample.
6. The method of claim 1 , wherein the subjecting the nucleic acids step is a multiplex amplification for a plurality of targets, and each additional second-stage amplification chamber comprises a pair of primers for one of the targets.
7. The method of claim 6 , wherein each pair of primers in the second-stage amplification chambers is configured to be nested within primers used in the subjecting the nucleic acids step.
8. The method of claim 1 wherein the second-stage amplification chambers are provided in an array and the performing step includes thermal cycling the array.
9. The method of claim 1 wherein the lysing step further includes contacting the sample with lysing particles.
10. The method of claim 9 wherein the lysing step includes repeatedly impacting an exterior surface of the container by way of a paddle to generate high velocity impacts between the sample and the lysing particles.
11. The method of claim 1 wherein one or more second-stage amplification chambers comprise a pair of primers for amplification of a control nucleic acid and the performing second-stage amplification step further comprises amplification of the control nucleic acid.
12. The method of claim 1 wherein replicate second-stage amplifications are performed in a plurality of second-stage amplification chambers.
13. The method of claim 1 wherein the first port is heat-sealed subsequent to injecting the sample.
14. The method of claim 1 wherein the performing second-stage amplification step further comprises detecting the nucleic acids in the sample.
15. The method of claim 14 wherein second-stage amplification is detected in real-time.
16. The method of claim 14 wherein detecting the nucleic acids comprises fluorescence monitoring of the second-stage amplification chambers during second-stage amplification.
17. The method of claim 16 wherein a computer is used to collect fluorescence data.
18. The method of claim 14 wherein second-stage amplification is detected through post-amplification analysis.
19. The method of claim 18 wherein the post-amplification analysis is a nucleic acid melting-curve analysis.
20. The method of claim 19 wherein detecting amplification comprises fluorescence monitoring of the second-stage amplification chambers during a post-amplification melting-curve analysis of the second-stage amplification products.
21. The method of claim 20 wherein a computer is used to collect fluorescence data.
22. The method of claim 1 further comprising
injecting a fluid into a second sealable port for hydrating reaction components, and
sealing the second port.
23. A method of amplifying nucleic acids in a sample, comprising the steps of
providing a container having
a plurality of fluidly connected reaction blisters including a lysis blister containing lysing particles, a nucleic acid preparation blister, and an amplification blister,
an array of second-stage amplification chambers fluidly connected to the amplification blister, each additional second-stage amplification chamber comprising primers for further amplifying a specific nucleic acid that may be present in the sample, and
one or more sealable ports fluidly connected to the reaction blisters, the sealable ports providing the only access from an exterior of the container to reaction blisters such that when all of the one or more sealable ports is sealed, the container is fully closed,
injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,
lysing cells in the lysis blister by generating high velocity impacts,
extracting the nucleic acids from the sample using magnetic beads, and moving the extracted nucleic acids to the amplification blister,
subjecting the nucleic acids in the amplification blister to amplification conditions,
fluidly moving a portion of the nucleic acids to each of the additional second-stage amplification chambers in the array, and
performing second-stage amplification in the additional second-stage amplification chambers.
24. The method of claim 1 , further comprising the step of releasing the nucleic acids from the magnetic beads prior to moving the nucleic acids to the amplification blister.