Self-contained biological analysis

• US 8,394,608 B2
• Filed: 05/08/2006
• Issued: 03/12/2013
• Est. Priority Date: 05/09/2005
• Status: Active Grant

• Current Assignee: BioFire Diagnostics Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: BioFire Diagnostics Incorporated (Compagnie Merieux Alliance SAS)
• Inventors: Ririe, Kirk M., Newswander, Michael R., Rasmussen, Randy P., Poritz, Mark Aaron, Smith, Stewart Benjamin, Jones, David E., Kessler, Gary Clark
• Primary Examiner(s): Strzelecka, Teresa E
• Assistant Examiner(s): Pande, Suchira
• Application Number:
  US11/913,120
• Publication Number:
  US 20100105029A1
• Time in Patent Office: 2,500 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 2400/065 sliding valves
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2523/303 : Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device

Abstract

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.

Claims

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.

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.

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.