Automated process for isolating and amplifying a target nucleic acid sequence

• US 6,335,166 B1
• Filed: 04/30/1999
• Issued: 01/01/2002
• Est. Priority Date: 05/01/1998
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Horner, Glenn A., Smith, Robert J., Burns, Ralph E., Schneider, Robert E., Ammann, Kelly G., Hansberry, Ernest V., Nieglos, Donald J., Kling, John E., Jakub, Cheryl A.
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US09/303,030
• Time in Patent Office: 977 Days
• Field of Search: 435/91.2, 435/6
• US Class Current: 435/6.11
• 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 or biological 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 analyzer for performing multiple diagnostic assays simultaneously includes multiple stations, or modules, in which discrete aspects of the assay are performed on fluid samples contained in reaction receptacles. The analyzer includes stations for automatically preparing a specimen sample, incubating the sample at prescribed temperatures for prescribed periods, preforming an analyte isolation procedure, and ascertaining the presence of a target analyte. An automated receptacle transporting system moves the reaction receptacles from one station to the next. The analyzer further includes devices for carrying a plurality of specimen tubes and disposable pipette tips in a machine-accessible manner, a device for agitating containers of target capture reagents comprising suspensions of solid support material and for presenting the containers for machine access thereto, and a device for holding containers of reagents in a temperature controlled environment and presenting the containers for machine access thereto. A method for performing an automated diagnostic assay includes an automated process for isolating and amplifying a target analyte. The process is performed by automatically moving each of a plurality of reaction receptacles containing a solid support material and a fluid sample between stations for incubating the contents of the reaction receptacle and for separating the target analyte bound to the solid support from the fluid sample. An amplification reagent is added to the separated analyte after the analyte separation step and before a final incubation step.

Claims

1. An automated process for isolating and amplifying a target nucleic acid sequence that may be present in a fluid sample contained in a reaction receptacle, said process comprising:

a) separating the target sequence from non-target nucleic acid that may be present in the fluid sample in a separation station;

b) transporting the reaction receptacle containing the separated target sequence, if present in the fluid sample, from the separation station to an amplifying incubation station comprising one or more incubators, each said incubator of said amplifying incubation station defining a temperature-controlled chamber; and

c) incubating the contents of the reaction receptacle, to which one or more amplification reagents have been provided, in said amplifying incubation station for a period of time and under conditions sufficient to permit the target sequence to be amplified,

wherein each of steps a)-c) is automated.

2. The automated process of claim 1, further comprising:

transporting the reaction receptacle to an immobilizing incubation station comprising one or more incubators, each said incubator of said immobilizing incubation station defining a temperature-controlled chamber;

incubating the contents of the reaction receptacle, to which a solid support material has been provided, for a period of time and under conditions sufficient to permit the target sequence to be immobilized by the solid support material;

transporting the reaction receptacle to said separation station, wherein the separating step includes separating the target sequence from the non-target nucleic acid that may be present in the fluid sample within said separation station by a separation procedure that includes isolating the solid support material within the reaction receptacle; and

removing the fluid sample from the reaction receptacle.

3. The automated process of claim 2, wherein said immobilizing and amplifying incubation stations are independent of one another or share at least one incubator in common.

4. The automated process of claim 2, wherein said immobilizing and amplifying incubation stations are independent of one another.

5. The automated process of claim 2, wherein the reaction receptacle is transported to said immobilizing incubation station from a holding station for holding a plurality of reaction receptacles.

6. The automated process of claim 5, wherein the step of transporting the reaction receptacle to said immobilizing incubation station is automated.

7. The automated process of claim 2, wherein the solid support material comprises a magnetically responsive particle having a polynucleotide bound thereto.

8. The automated process of claim 7, wherein a target nucleic acid containing the target sequence is hybridized to a capture probe prior to being immobilized by the solid support material.

9. The automated process of claim 7, wherein the contents of the reaction receptacle are subjected to a magnetic field during said separation procedure.

10. The automated process of claim 2, wherein the fluid sample is aspirated from the reaction receptacle after isolating the solid support material.

11. The automated process of claim 10, further comprising:

providing a wash buffer to the reaction receptacle after removing the fluid sample from the reaction receptacle, and thereafter;

agitating the reaction receptacle to resuspend the solid support material;

isolating the solid support within the reaction receptacle; and

removing the wash buffer from the reaction receptacle.

12. The automated process of claim 2, wherein the incubators of said amplifying incubation station are maintained at a temperature or temperatures different than the temperature or temperatures maintained by the incubators of said immobilizing incubation station.

13. The automated process of claim 2, further comprising raising or lowering the temperature of the contents of the receptacle prior to transporting the reaction receptacle to at least one of said immobilizing and hybridizing incubation stations.

14. The automated process of claim 2, wherein the solid support material is provided to the reaction prior to transporting the reaction receptacle to said immobilizing incubation station.

15. The automated process of claim 2, further comprising:

transporting the reaction receptacle to a hybridizing incubation station comprising one or more incubators, each said incubator of said hybridizing incubation station defining a temperature-controlled chamber; and

incubating, within said hybridizing incubation station, the contents of the reaction receptacle, to which one or more probe reagents have been provided, for a period of time and under conditions sufficient to permit the probe to hybridize to the target sequence or an amplicon thereof.

16. The automated process of claim 15, further comprising detecting the presence or absence of the probe hybridized to the target sequence, or an amplicon thereof, as an indication of the presence or absence of an organism or virus or one or more members of a group of organisms or viruses in the fluid sample.

17. The automated process of claim 16, further comprising transporting the reaction receptacle to a detection station for detecting the presence or amount of the label in the reaction receptacle.

18. The automated process of claim 17, wherein the step of transporting the reaction receptacle to said detection station is automated.

19. The automated process of claim 15, wherein the step of transporting the reaction receptacle to said hybridizing incubation station is automated.

20. The automated process of claim 2, wherein the step of transporting the reaction receptacle to said separation station is automated.

21. The automated process of claim 20, wherein the step of transporting the reaction to said immobilizing incubation station is automated.

22. The automated process of claim 1, further comprising:

transporting the reaction receptacle to a hybridizing incubation station comprising one or more incubators, each said incubator of said hybridizing incubation station defining a temperature-controlled chamber; and

incubating, within said hybridizing incubation station, the contents of the reaction receptacle, to which one or more probe reagents have been provided, for a period of time and under conditions sufficient to permit the probe to hybridize to the target sequence or an amplicon thereof.

23. The automated process of claim 22, wherein said amplifying and hybridizing incubation stations are independent of one another or share at least one incubator in common.

24. The automated process of claim 22, wherein said amplifying and hybridizing incubation stations are independent of one another.

25. The automated process of claim 22, wherein the probe reagents include a probe having a detectable label.

26. The automated process of claim 25, wherein the label is a fluorescent dye or chemiluminescent compound.

27. The automated process of claim 25, further comprising detecting the presence or absence of the probe hybridized to the target sequence, or an amplicon thereof, by detecting the amount of light emitted by the contents of the reaction receptacle, Thereby providing an indication of the presence or absence of an organism or virus or one or more members of a group of organisms or viruses in the fluid sample.

28. The automated process of claim 27, further comprising determining the amount of the organism or virus or the organisms or viruses in the fluid sample.

29. The automated process of claim 27, further comprising transporting each reaction receptacle to a luminometer station for detecting the amount of light emitted by the contents of the reaction receptacle.

30. The automated process of claim 29, wherein the step of transporting the receptacle to said luminometer station is automated.

31. The automated process of claim 25, further comprising transporting the reaction receptacle to a detection station for detecting the presence or amount of the label in the reaction receptacle.

32. The automated process of claim 31, wherein said detection station comprises a luminometer for detecting the amount of light emitted by the contents of the reaction receptacle.

33. The automated process of claim 31, wherein the step of transporting the receptacle to said detection station is automated.

34. The automated process of claim 22, further comprising detecting the presence or absence of the probe hybridized to the target sequence, or an amplicon thereof, as an indication of the presence or absence of an organism or virus or one or more members of a group of organisms or viruses in the fluid sample.

35. The automated process of claim 34, further comprising determining the amount of the organism or virus or the organisms or viruses in the fluid sample.

36. The automated process of claim 34, further comprising transporting the reaction receptacle to a deactivation station and providing to the reaction receptacle a deactivating reagent after permitting the target sequence to be amplified.

37. The automated process of claim 22, wherein the one or more probe reagents are provided to the reaction receptacle after transporting the reaction receptacle to said hybridizing incubation station.

38. The automated process of claim 22, wherein the step of transporting the reaction receptacle to said hybridizing incubation station is automated.

39. The automated process of claim 1, further comprising raising or lowering the temperature of the contents of the reaction receptacle prior to transporting the reaction receptacle to said amplifying incubation station.

40. The automated process of claim 1, wherein the target sequence is amplified by a polymerase chain reaction procedure.

41. The automated process of claim 1, wherein the target sequence is amplified by a transcription-mediated amplification procedure.

42. The automated process of claim 1, wherein the one or more amplification reagents are provided to the reaction receptacle prior to transporting the reaction receptacle to said amplifying incubation station.

43. The automated process of claim 1, further comprising dispensing a fluid sample into the reaction receptacle.

44. The automated process of claim 1, further comprising transporting the reaction receptacle to said amplifying incubation station by means of a receptacle transport mechanism.

45. The automated process of claim 1, wherein the reaction receptacle comprises an integrally-formed array of a plurality of test tubes so that a target nucleic acid sequence that may be present in a fluid sample contained in each test tube of the reaction receptacle is isolated and amplified during said automated process.

46. The automated process of claim 1, further comprising transporting the reaction receptacle to a deactivation station and providing to the reaction receptacle a deactivating reagent after permitting the target sequence to be amplified.

47. The automated process of claim 46, wherein the deactivating reagent comprises a chemical agent selected from the group consisting of solutions of potassium permanganate, formic acid, solutions of sodium hypochlorite, hydrazine, and dimethyl sulfate.

48. The automated process of claim 46, wherein the step of transporting the reaction receptacle to said deactivation station is automated.