Method for detecting the presence of a nucleic acid in a sample

• US 9,150,908 B2
• Filed: 05/21/2014
• Issued: 10/06/2015
• Est. Priority Date: 05/01/1998
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Ammann, Kelly G., Burns, Ralph E., Hansberry, Ernest V., Horner, Glenn A., Jakub, Cheryl A., Nieglos, Donald J., Schneider, Robert E., Smith, Robert J., Kling, John E.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/284,025
• Publication Number:
  US 20140248619A1
• Time in Patent Office: 503 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/91.51, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 1/1
• CPC Class Codes: B01F 29/10 with receptacles rotated about two different axes, e.g. receptacles having planetary motion
• CPC Class Codes: B01F 29/30 Mixing the contents of individual packages or containers, e.g. by rotating tins or bottles
• CPC Class Codes: B01F 29/322 of two or more containers supported for simultaneous mixing, e.g. for bottles in crates
• CPC Class Codes: B01F 29/40354 arranged for planetary motion
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• 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: B01L 7/5255 by moving sample containers
• CPC Class Codes: B01L 9/06 Test-tube stands; Test-tube holders
• CPC Class Codes: B03C 1/282 with associated accumulation indicator, e.g. Hall sensor
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/10 the purpose or use of
• CPC Class Codes: G01N 2035/00356 Holding samples at elevated temperature (incubation)
• CPC Class Codes: G01N 2035/00524 Mixing by agitating sample carrier
• CPC Class Codes: G01N 2035/0437 : Cleaning cuvettes or reaction vessels
• CPC Class Codes: G01N 2035/0455 : Coaxial carousels
• CPC Class Codes: G01N 2035/103 : using disposable tips
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: G01N 35/0099 : comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/025 : having a carousel or turntable for reaction cells or cuvettes
• CPC Class Codes: G01N 35/1065 : Multiple transfer devices
• CPC Class Codes: Y10T 156/1105 : Delaminating process responsive to feed or shape at delamination
• CPC Class Codes: Y10T 156/1906 : Delaminating means responsive to feed or shape at delamination
• CPC Class Codes: Y10T 29/53657 : to apply or remove a resilient article [e.g., tube, sleeve, etc.]
• CPC Class Codes: Y10T 29/53683 : Spreading parts apart or separating them from face to face engagement
• CPC Class Codes: Y10T 29/53843 : Tube, sleeve, or ferrule inserting or removing
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/111666 : Utilizing a centrifuge or compartmented rotor
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/114998 : with treatment or replacement of aspirator element [e.g., cleaning, etc.]
• CPC Class Codes: Y10T 436/119163 : with aspirator of claimed structure
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

An automated method for detecting the presence of a nucleic acid in a sample, where the method is performed within a housing of a self-contained, stand-alone analyzer. The method includes purifying the nucleic acid after it has been immobilized on a magnetically-responsive solid support. A pipette of the analyzer is used to form a reaction mixture comprising the purified nucleic acid and all reagents required to perform a nucleic acid amplification. Amplification products are synthesized that include a nucleotide sequence contained in the nucleic acid or the complement of the nucleic acid. The amplification products are exposed to a probe in a mixture, where the probe forms a hybrid with one of the amplification products. The formation of the hybrid in the mixture provides an indication of the presence of the nucleic acid in the sample.

Claims

1. A method for detecting the presence of a nucleic acid in a sample, the method comprising performing within a housing of a self-contained, stand-alone analyzer the automated steps of:

a) contacting the sample with a solid support such that a complex comprising the nucleic acid and the solid support is formed in the sample, wherein the solid support comprises a magnetically-responsive particle, and wherein the complex is suspended in a fluid component of the sample;

b) after step a), subjecting the complex contained in the sample to a magnetic field;

c) while the complex contained in the sample is subjected to the magnetic field, aspirating at least a portion of the fluid component of the sample from the complex;

d) after step c), washing the solid support one or more times with a wash buffer, thereby providing a purified form of the nucleic acid;

e) forming a reaction mixture with a pipette of the analyzer, wherein the reaction mixture comprises the purified form of the nucleic acid and all reagents required to perform a nucleic acid amplification;

f) synthesizing amplification products in the reaction mixture, each of the amplification products comprising a nucleotide sequence contained in the nucleic acid or its complement;

g) exposing an amplification product that is one of the amplification products synthesized in step f) to a probe having a label, such that a hybrid comprising the probe and the amplification product is formed in solution in a mixture containing the probe and the amplification products; and

h) in the mixture of step g), detecting the label after formation of the hybrid, wherein the formation of the hybrid in the mixture is an indication of the presence of the nucleic acid in the sample,

wherein steps b)-d) are performed at a first station of the analyzer and step f) is performed at a second station of the analyzer, and

wherein step f) is performed within an enclosure having a receptacle access opening, the receptacle access opening being closed by a door of the enclosure during step f).

2. The method of claim 1, further comprising, prior to step a), a manual step of providing the sample and the reagents to the analyzer, wherein the sample and the reagents are separately provided to the analyzer.

3. The method of claim 1, wherein the complex formed in step a) further comprises a capture probe hybridized to the nucleic acid.

4. The method of claim 1, wherein the fluid component is aspirated through a tiplet in frictional engagement with an aspirator tube of a fluid aspirator of the analyzer.

5. The method of claim 1, further comprising, prior to step c), an automated step of sensing the level of the fluid component.

6. The method of claim 1, wherein the portion of the fluid component aspirated in step c) is drawn through an aspirator tube to a fluid waste container.

7. The method of claim 6, wherein the fluid waste container is situated in a lower chassis of the analyzer beneath a processing deck contained within the housing.

8. The method of claim 1, wherein the steps of the method are performed in a single receptacle.

9. The method of claim 8, wherein the receptacle is a cylindrical tube.

10. The method of claim 8, wherein the receptacle is one of a plurality of integrally formed receptacles.

11. The method of claim 1, wherein the amplification products formed in step f) comprise amplification products having the nucleotide sequence contained in the nucleic acid and amplification products having the nucleotide sequence contained in the complement of the nucleic acid.

12. The method of claim 1, wherein the probe hybridizes with specificity to the amplification product in step g), and wherein the nucleic acid is from a disease-associated pathogen.

13. The method of claim 1, wherein the label is a fluorescent or chemiluminescent label.

14. The method of claim 1, wherein the first and second stations are situated on a processing deck contained within the housing.

15. The method of claim 1, wherein steps f) and g) are performed at separate stations situated within the housing.

16. The method of claim 15, wherein step f) is performed in a first incubator and step g) is performed in a second incubator, and wherein the first and second incubators are in a laterally spaced-apart relationship.

17. The method of claim 15, wherein the stations are situated on a processing deck contained within the housing.

18. The method of claim 1, wherein the analyzer is controlled by a computer controller that is integrally housed within the analyzer.

19. The method of claim 1, wherein the steps of the method are performed at multiple stations situated on a processing deck contained within the housing.

20. The method of claim 1, wherein the housing remains closed when the steps of the method are performed.

21. The method of claim 1, wherein step f) is performed at a substantially uniform temperature.