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

• US 8,546,110 B2
• Filed: 09/30/2008
• Issued: 10/01/2013
• 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., Kling, John E., Nieglos, Donald J., Schneider, Robert E., Smith, Robert J.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US12/241,353
• Publication Number:
  US 20090029352A1
• Time in Patent Office: 1,827 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/6.1, 435/6.12, 435/6.11, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• 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 preparing and amplifying a sequence contained in a nucleic acid present in a sample, the nucleic acid being prepared in a receptacle that is part of a unit that includes a plurality of receptacles and holds a removable contact-limiting element for aspirating a fluid component of the sample from the receptacle.

Claims

1. A method for detecting the presence of a nucleic acid in a sample, the method comprising performing in an analyzer the automated steps of:

(a) combining, in a receptacle, a sample containing a nucleic acid and a solid support comprising a magnetically-responsive particle, the solid support forming a complex with the nucleic acid, wherein the complex is suspended in a fluid component of the sample, the receptacle being part of a unit comprising a plurality of integrally formed receptacles, and the unit holding a removable contamination-limiting element configured to permit passage of the fluid component of the sample therethrough;

(b) subjecting the contents of the receptacle to a magnetic field, thereby isolating the nucleic acid within the receptacle;

(c) mounting the contamination-limiting element onto a fluid aspirator;

(d) purifying the nucleic acid isolated in step (b) by contacting the fluid component of the sample with the contamination-limiting element and aspirating at least a portion of the fluid component with the fluid aspirator while the complex remains subjected to the magnetic field;

(e) forming a reaction mixture comprising the purified nucleic acid of step (d) and all reagents required to perform a nucleic acid amplification;

(f) performing a nucleic acid amplification with the reaction mixture formed in step (e), the nucleic acid amplification resulting in the enzymatic synthesis of amplification products, each of the amplification products comprising (i) a target sequence contained in the nucleic acid or (ii) the complement of the target sequence;

(g) selectively hybridizing a probe having a label to the target sequence or its complement of an amplification product that is one of the amplification products synthesized in step (f), thereby forming a hybrid in a mixture that comprises the probe and the amplification product; and

(h) in the mixture, detecting the 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.

2. The method of claim 1, wherein each of the receptacles comprises a cylindrical tube.

3. The method of claim 1, further comprising performing in the analyzer an automated step of sensing the level of the fluid component in step (d), wherein the contamination-limiting element is mounted on an aspirator tube of the fluid aspirator, and wherein the aspirator tube and the contamination-limiting element constitute a portion of a capacitor for performing capacitive fluid level sensing.

4. The method of claim 1, wherein the contamination-limiting element is one of a plurality of contamination-limiting elements held by the unit, wherein each of the contamination-limiting elements is configured to permit passage of a fluid component of a sample therethrough, and wherein each of the contamination-limiting elements is operationally associated with one of the receptacles of the unit.

5. The method of claim 1, wherein the contamination-limiting element is held in a frictional fit by the unit when the contamination-limiting element is mounted onto the fluid aspirator in step (c).

6. The method of claim 1, wherein the complex further comprises a capture probe, and wherein the capture probe is immobilized on the solid support and hybridized to the nucleic acid.

7. The method of claim 1, further comprising performing in the analyzer an automated step of washing the solid support one or more times with a wash buffer after step (d) and prior to step (e).

8. The method of claim 1, wherein each of the amplification products consists of a nucleic acid sequence having the target sequence or its complement.

9. The method of claim 1, wherein the amplification products include nucleic acid sequences having a sequence that is the same as the target sequence and nucleic acid sequences having a sequence that is the complement of the target sequence.

10. The method of claim 1, wherein step (f) is carried out in a temperature-controlled incubator maintained at a substantially uniform temperature.

11. The method of claim 1, wherein step (f) is performed in a temperature-controlled chamber of a first incubator, and wherein step (g) is performed in a temperature-controlled chamber of a second incubator, the first and second incubators being in a spaced-apart relationship.

12. The method of claim 1, wherein the hybrid is in solution.

13. The method of claim 1, wherein the steps of the method are performed within a housing of the analyzer.

14. The method of claim 13, wherein the steps of the method are performed at multiple locations of a processing deck contained within the housing.

15. The method of claim 13, wherein the housing remains closed during the method.

16. A method for detecting the presence of a nucleic acid in a sample, the method comprising performing in an analyzer the automated steps of:

(a) combining, in a receptacle, a sample containing a nucleic acid and a solid support comprising a magnetically-responsive particle, the solid support forming a complex with the nucleic acid, wherein the complex is suspended in a fluid component of the sample, the receptacle being part of a unit comprising a plurality of integrally formed receptacles, and the unit holding a removable tiplet comprising an axially extending through-hole for drawing fluids therethrough;

(b) subjecting the contents of the receptacle to a magnetic field, thereby isolating the nucleic acid within the receptacle;

(c) mounting the tiplet onto a fluid aspirator;

(d) purifying the nucleic acid isolated in step (b) by contacting the fluid component of the sample with the tiplet and aspirating at least a portion of the fluid component with the fluid aspirator while the complex remains subjected to the magnetic field;

(e) forming a reaction mixture comprising the purified nucleic acid of step (d) and all reagents required to perform a nucleic acid amplification;

(f) performing a nucleic acid amplification with the reaction mixture formed in step (e), the nucleic acid amplification resulting in the enzymatic synthesis of amplification products, each of the amplification products comprising (i) a target sequence contained in the nucleic acid or (ii) the complement of the target sequence;

(g) selectively hybridizing a probe having a label to the target sequence or its complement of an amplification product that is one of the amplification products synthesized in step (f), thereby forming a hybrid in a mixture that comprises the probe and the amplification product; and

in the mixture, detecting the formation of the hybrid, wherein the formation of the hubrid in the mixture is an indication of the presence of the nucleic acid in the sample.

17. The method of claim 16, wherein each of the receptacles comprises a cylindrical tube.

18. The method of claim 16, wherein the tiplet is electrically conductive.

19. The method of claim 18, further comprising performing in the analyzer an automated step of sensing the level of the fluid component in step (d), wherein the tiplet is mounted on an aspirator tube of the fluid aspirator, and wherein the aspirator tube and the tiplet constitute a portion of a capacitor for performing capacitive fluid level sensing.

20. The method of claim 16, wherein the tiplet is one of a plurality of tiplets held by the unit, wherein each of the tiplets is configured to permit passage of a fluid component of a sample therethrough, and wherein each of the tiplets is operationally associated with one of the receptacles of the unit.

21. The method of claim 16, wherein the tiplet is held in a frictional fit by the unit when the tiplet is mounted onto the fluid aspirator in step (c).

22. The method of claim 16, wherein the complex further comprises a capture probe, and wherein the capture probe is immobilized on the solid support and hybridized to the nucleic acid.

23. The method of claim 16, further comprising performing in the analyzer an automated step of washing the solid support one or more times with a wash buffer after step (d) and prior to step (e).

24. The method of claim 16, wherein each of the amplification products consists of a nucleic acid sequence having the target sequence or its complement.

25. The method of claim 16, wherein the amplification products include nucleic acid sequences having a sequence that is the same as the target sequence and nucleic acid sequences having a sequence that is the complement of the target sequence.

26. The method of claim 16, wherein step (f) is carried out in a temperature-controlled incubator maintained at a substantially uniform temperature.

27. The method of claim 16, wherein step (f) is performed in a temperature-controlled chamber of a first incubator, and wherein step (g) is performed in a temperature-controlled chamber of a second incubator, the first and second incubators being in a spaced-apart relationship.

28. The method of claim 16, wherein the hybrid is in solution.

29. The method of claim 16, wherein the steps of the method are performed within a housing of the analyzer.

30. The method of claim 29, wherein the steps of the method are performed at multiple locations of a processing deck contained within the housing.

31. The method of claim 29, wherein the housing remains closed during the method.