Method for separating an analyte from a sample

• US 8,247,176 B2
• Filed: 03/07/2011
• Issued: 08/21/2012
• Est. Priority Date: 12/24/1998
• Status: Expired due to Fees

• Current Assignee: Cepheid (Danaher Corporation)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Petersen, Kurt E., McMillan, William A., Christel, Lee A., Chang, Ronald, Pourahmadi, Farzad, Ching, Jesus, Kovacs, Gregory T. A., Northrup, M. Allen
• Primary Examiner(s): Beisner, William H
• Application Number:
  US13/042,203
• Publication Number:
  US 20110165562A1
• Time in Patent Office: 533 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: B01D 15/00 Separating processes involving the treatment of liquids with solid sorbents using liquid sorbents B01D11/00; ion exchange processes or materials, sorbent materials in general B01J, e.g. sorbents for chromatography B01J20/281; for investigating or analysing materials G01N30/00; Apparatus therefor
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0644 rotary valves
• 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: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

An analyte is separated from a fluid sample by introducing the sample into a cartridge having a sample port and a first flow path extending from the sample port. The first flow path includes an extraction chamber containing a solid support for capturing the analyte from the sample. The cartridge has a second flow path for eluting the captured analyte from the extraction chamber, the second flow diverging from the first flow path after passing through the extraction chamber. The sample is forced to flow through the extraction chamber and into a waste chamber, thereby capturing the analyte with the solid support as the sample flows through the extraction chamber. The captured analyte is then eluted from the extraction chamber by forcing an elution fluid to flow through the extraction chamber and along the second flow path.

Claims

1. A method for extracting nucleic acid from a sample, the sample containing cells, spores, or microorganisms, the method comprising the steps of:

a) introducing the sample into a cartridge having;

i) a lysing chamber for lysing the cells, spores, or microorganisms to release the nucleic acid therefrom;

ii) a desiccant adjacent to the lysing chamber;

iii) a capture region containing capture material for capturing the nucleic acid;

iv) at least one waste chamber; and

v) at least a third chamber for receiving the nucleic acid extracted from the sample;

b) contacting the sample with paper or membrane material in the lysing chamber, the paper or membrane material being impregnated with at least one chemical for lysing the cells, spores, or microorganisms in the sample, while drying the sample on the paper or the membrane material by heating and absorbing moisture with the desiccant;

c) lysing the cells, spores, or microorganisms with the chemical to release the nucleic acid from the cells, spores, or microorganisms, while binding contaminants or inhibitors in the sample to the paper or membrane material;

d) removing the nucleic acid from the lysing chamber by placing fluid in the lysing chamber, releasing the nucleic acid from the paper or membrane material into the fluid while the contaminants or inhibitors remain bound to the paper or membrane material, and forcing the fluid containing the nucleic acid to flow out of the lysing chamber and through the capture region, thereby capturing the nucleic acid from the fluid with the capture material in the capture region;

e) forcing the fluid that has flowed through the capture region to flow into the waste chamber; and

f) eluting the captured nucleic acid from the capture region and forcing the eluted nucleic acid to flow into the third chamber.

2. The method of claim 1, the third chamber comprises a reaction chamber, and the method further comprises the steps of:

i) amplifying the nucleic acid in the reaction chamber; and

ii) detecting the amplified nucleic acid.

3. The method of claim 2, wherein the amplification requires temperature control of the reaction chamber, the portion of the cartridge defining the reaction chamber protrudes from the rest of the cartridge body, and the temperature of the reaction chamber is controlled by inserting the reaction chamber into a thermal sleeve and heating or cooling the reaction chamber according to a time/temperature profile.

4. The method of claim 2, wherein the cartridge further includes a reagent chamber containing dried or lyophilized reagents, and the method further comprises the step of mixing the eluted nucleic acid with the reagents in the reagent chamber prior to forcing the nucleic acid to flow into the reaction chamber.

5. The method of claim 1, wherein the capture material comprises at least one solid support selected from the group consisting of filters, membranes, beads, fiber, glass wool, filter paper, polymers, and gel.

6. The method of claim 1, wherein the capture region comprises an extraction chamber formed in a microfluidic chip, and wherein the capture material comprises an array of microstructures extending into the extraction chamber, each of the microstructures having an aspect ratio (height to width) of at least 2:

1.

7. The method of claim 1, wherein the captured nucleic acid is eluted from the capture region by forcing elution fluid to flow through the capture region, and wherein the volume of sample placed in the lysing chamber is greater than the volume of elution fluid forced to flow through the capture region, whereby the nucleic acid extracted from the sample is concentrated in the smaller volume of elution fluid.

8. The method of claim 1, wherein the ratio of the volume of fluid forced to flow through the capture region in step (d) to the volume capacity of the capture region is at least 2:

1.

9. The method of claim 1, wherein the nucleic acid is released from the paper or membrane material into the fluid in the lysing chamber by heating the paper or membrane material.

10. The method of claim 1, wherein step (d) is preceded by the additional steps of binding the nucleic acid released from the cells, spores, or microorganisms to the paper or membrane material, washing the lysing chamber with wash fluid, and forcing the wash fluid to flow out of the lysing chamber and into the at least one waste chamber while the nucleic acid remains bound to the paper or membrane material.

11. The method of claim 1, wherein the chemical comprises at least one lysing agent selected from the group consisting of enzymes, detergents, and chaotropes.

12. The method of claim 1, wherein the chemical comprises a chaotropic salt.

13. The method of claim 1, wherein the paper or membrane material comprises cellulose, nitrocellulose, polycarbonate, or nylon.