Method for analyzing a fluid sample

• US 6,881,541 B2
• Filed: 03/06/2001
• Issued: 04/19/2005
• Est. Priority Date: 05/28/1999
• Status: Expired due to Term

• Current Assignee: Cepheid (Danaher Corporation)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Chang, Ronald, Petersen, Kurt E., Northrup, M. Allen, Belgrader, Phillip, McMillan, William A., Pourahmadi, Farzad, Ching, Jesus, Dority, Douglas B., Sakai, Stanley H., Taylor, Michael T.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US09/800,590
• Publication Number:
  US 20010012612A1
• Time in Patent Office: 1,505 Days
• Field of Search: 435/6, 435/259, 435/91.2, 435/287.2, 435/288.5, 435/288.6, 435/306.1, 241/2, 536/25.4, 536/25.41, 436/518, 436/178, 436/531
• US Class Current: 435/6.11
• 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 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• 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: C12N 1/066 by physical methods
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10T 137/87716 For valve having a flexible diaphragm valving member
• CPC Class Codes: Y10T 137/88046 Biased valve with external operator
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A method for extracting nucleic acid from a fluid sample comprises the steps of introducing the sample into a cartridge having a sample flow path and a lysing chamber in the sample flow path. The lysing chamber contains at least one filter for separating cells or viruses from the sample. The sample is forced to flow through the lysing chamber to capture the cells or viruses with the filter, while used sample fluid flows to waste. The captured cells or viruses are disrupted to release their nucleic acid, the nucleic acid is eluted from the lysing chamber, and optionally the nucleic acid is amplified and detected in a reaction chamber of the cartridge.

Claims

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

a) introducing the sample into a cartridge having;

i) a lysing chamber for lysing the cells or viruses to release the nucleic acid therefrom, wherein the lysing chamber contains at least one filter having a pore size sufficient to capture the cells or viruses in the sample as the sample flows through the lysing chamber, and wherein the lysing chamber further contains beads for rupturing the cells or viruses;

b) a waste chamber for receiving used sample fluid that has flowed through the lysing chamber; and

c) at least a third chamber for receiving the nucleic released from the cells or viruses;

b) forcing the sample to flow through the lysing chamber to capture the cells or viruses with the filter;

c) forcing the used sample fluid that has flowed through the lysing chamber to flow into the waste chamber;

d) placing a lysis buffer in the lysing chamber;

e) agitating the beads to lyse the cells or viruses, wherein the beads are agitated by sonicating the lysing chamber using an ultrasonic transducer coupled to a wall of the lysing chamber; and

f) forcing the nucleic acid released from the cells or viruses to flow into the third chamber.

2. The method of claim 1, further comprising the step of mixing the nucleic acid with reagents in the third chamber.

3. The method of claim 2, wherein the cartridge further includes a reaction chamber, and the method further comprises the steps of forcing the nucleic acid and reagents to flow into the reaction chamber and amplifying the nucleic acid in the reaction chamber.

4. The method of claim 3, further comprising the step of detecting the amplified nucleic acid in the reaction chamber.

5. The method of claim 1, wherein the third chamber is a reaction chamber and the method further comprises the step of amplifying the nucleic acid in the reaction chamber.

6. The method of claim 5, further comprising the step of detecting the amplified nucleic acid in the reaction chamber.

7. The method of claim 1, wherein the ratio of the volume of sample forced to flow through the lysing chamber to the volume capacity of the lysing chamber is at least 2:

1, and wherein the volume of sample forced to flow through the lysing chamber is at least 100 microliters.

8. The method of claim 1, wherein the ratio of the volume of sample forced to flow through the lysing chamber to the volume capacity of the lysing chamber is at least 5:

1.

9. The method of claim 1, wherein the volume of sample forced to flow through the lysing chamber is at least 1 ml.

10. The method of claim 1, wherein the beads have a binding affinity for the cells or viruses to be disrupted, and wherein the method further comprises the step of binding the cells or viruses to the beads.

11. The method of claim 1, wherein the beads have a binding affinity for the nucleic acid, and wherein the method further comprises the step of binding the nucleic acid to the beads.

12. The method of claim 1, wherein the lysing chamber contains a first set of beads for binding the cells or viruses and a second set of beads for rupturing the cells or viruses, the method further comprises the step of binding the cells or viruses to the first set of beads, and the step of disrupting the cells or viruses comprises rupturing the cells or viruses with the second set of beads.

13. The method of claim 1, further comprising the step of sonicating the lysing chamber while forcing the sample to flow through the lysing chamber.

14. The method of claim 1, further comprising the step of forcing a wash fluid to flow through the lysing chamber after forcing the sample to flow through the lysing chamber and prior to placing the lysis buffer in the lysing chamber.

15. The method of claim 1, wherein the lysis buffer comprises a lysing reagent.

16. The method of claim 1, wherein the cartridge has a first filter for filtering coarse material from the sample and a second filter in the lysing chamber for separating the cells or viruses from the sample, the second filter having a smaller average pore size than the first filter, and wherein the method further comprises the step of forcing the sample to flow through the first filter, thereby filtering the coarse material from the sample, prior to forcing the sample to flow through the second filter.