Method for performing multi-formatted assays

• US 20060204997A1
• Filed: 03/10/2006
• Published: 09/14/2006
• Est. Priority Date: 03/10/2005
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Li, Haitao, Nguyen, Thanh N., Heinz, Robert E., Scalese, Robert F., Davis, Christopher B., Knight, Byron J., Macioszek, Jerzy A., Li, Florence F., Lair, Gary D.
• Granted Patent: US 7,897,337 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2035/00346 Heating or cooling arrangements
• 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 2035/0097 monitoring reactions as a function of time
• CPC Class Codes: G01N 2035/0441 for samples
• CPC Class Codes: G01N 2035/0443 for reagents
• CPC Class Codes: G01N 2035/0444 for cuvettes or reaction vessels
• CPC Class Codes: G01N 2035/0455 Coaxial carousels
• CPC Class Codes: G01N 2035/0491 Position sensing, encoding; closed-loop control
• CPC Class Codes: G01N 2035/103 using disposable tips
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6454 : using an integrated detector array
• CPC Class Codes: G01N 21/76 : Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 2201/0415 : Carrusel, sequential
• CPC Class Codes: G01N 35/00693 : Calibration
• CPC Class Codes: G01N 35/0092 : Scheduling
• 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 35/026 : having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: G01N 35/028 : having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/04 : Details of the conveyor system G01N35/021 - G01N35/028 take precedence
• CPC Class Codes: G01N 35/1002 : Reagent dispensers
• CPC Class Codes: Y10S 435/808 : Optical sensing apparatus
• CPC Class Codes: Y10S 435/809 : Incubators or racks or holders for culture plates or containers
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

An automated analyzer for performing multiple diagnostic assays simultaneously includes multiple stations in which discrete aspects of the assay are performed on fluid samples contained in sample vessels. The analyzer includes stations for automatically preparing a sample, incubating the sample, preforming an analyte isolation procedure, ascertaining the presence of a target analyte, and analyzing the amount of a target analyte. An automated receptacle transporting system moves the sample vessels from one station to the next. A method for performing an automated diagnostic assay includes an automated process for isolating and amplifying a target analyte, and, in one embodiment, a method for real-time monitoring of the amplification process.

Claims

1. A method for processing the contents of multiple reaction receptacles to quantitatively determine the amount of a first target nucleic acid present in a first portion of the reaction receptacles and to qualitatively determine the presence or absence of a second target nucleic acid present in a second portion of the reaction receptacles, said method comprising the automated steps of:

a) successively providing to a first heater a plurality of reaction receptacles, each said reaction receptacle being provided with amplification and enzyme reagents sufficient to amplify a first or second target sequence contained within a corresponding first or second target nucleic acid under the temperature conditions of said first heater;

b) successively and periodically moving reaction receptacles of a first portion of said reaction receptacles, to which detection reagents sufficient to detect a first amplification product containing said first target sequence or its complement have been provided, into operative proximity to a signal measuring device constructed and arranged to measure the amount of a signal emitted by the contents of a reaction receptacle placed in operative proximity to said signal measuring device and measuring the amount of signal associated with the presence of said first amplification product in each reaction receptacle of said first portion of reaction receptacles moved into operative proximity to said signal measuring device;

c) using a microprocessor to quantify the amount of said first target nucleic acid present in each reaction receptacle of said first portion of reaction receptacles based upon the measurements obtained in step b);

d) successively moving reaction receptacles of a second portion of said reaction receptacles, to which detection reagents sufficient to detect a second amplification product containing said second target sequence or its complement have been provided, into operative proximity to a signal detecting device constructed and arranged to detect the presence of a signal emitted by the contents of a reaction receptacle placed in operative proximity to said signal detecting device and detecting a signal associated with the presence of said second amplification product in each reaction receptacle of said second portion of reaction receptacles moved into operative proximity to said signal detecting device; and

f) using a microprocessor to determine the presence or absence of said second target nucleic acid in each reaction receptacle of said second portion of reaction receptacles based upon the measurements obtained in step d).

2. The method of claim 1, wherein said reaction receptacles are provided to said first heater using an automated reaction receptacle transport system.

3. The method of claim 2, wherein said transport system includes a first, rotatable transport mechanism for providing reaction receptacles to said first heater.

4. The method of claim 3, wherein said transport system includes a second, rotatable transport mechanism for removing reaction receptacles from said first heater.

5. The method of claim 2, wherein said transport system moves reaction receptacles of said second portion of reaction receptacles from said first heater into operative proximity to said signal detecting device in step d).

6. The method of claim 5 further comprising the automated step of moving reaction receptacles of said second portion of reaction receptacles from said first heater to a second heater, where said detection reagents are provided to reaction receptacles of said second portion of reaction receptacles under temperature conditions permitting a probe present in said detection reagents to detectably hybridize to said second target sequence or its complement.

7. The method of claim 6, wherein said second heater is an enclosed incubator having one or more doors for receiving and removing reaction receptacles from said incubator.

8. The method of claim 7, wherein said incubator includes a rotatable reaction receptacle carrier for receiving and moving reaction receptacles within said incubator.

9. The method of claim 6 further comprising the automated step of moving reaction receptacles of said second portion of reaction receptacles from said second heater into operative proximity to said signal detecting device.

10. The method of claim 9, wherein said signal detecting device is a luminometer and said probe is labeled with a chemiluminescent label.

11. The method of claim 1, wherein said first heater is an enclosed incubator having one or more doors for receiving and removing said reaction receptacles from said incubator.

12. The method of claim 11, wherein said incubator includes rotatable reaction receptacle carrier for receiving and moving reaction receptacles within said incubator.

13. The method of claim 1, wherein said amplification and enzyme reagents are provided to said reaction receptacles after said reaction receptacles have been provided to said first heater.

14. The method of claim 13, wherein said detection reagents are provided to reaction receptacles of said first portion of reaction receptacles after reaction receptacles of said first portion of reaction receptacles are provided to said first heater, and wherein reaction receptacles of said first portion of reaction receptacles are moved into operative proximity to said signal measuring device in step b) before being removed from said first heater.

15. The method of claim 14, wherein reaction receptacles of said second portion of reaction receptacles are removed from said first heater prior to being moved into operative proximity to said signal detecting device in step d), and wherein said detection reagents are provided to reaction receptacles of said second portion of reaction receptacles after being removed from said first heater.

16. The method of claim 1, wherein said amplification and enzyme reagents provided to reaction receptacles of said first portion of reaction receptacles in step a) include a primer or a promoter-primer, nucleoside triphosphates, and a nucleic acid polymerase capable of extending the 3′

end of said primer or promoter-primer hybridized to said first target nucleic acid under the temperature conditions of said first heater, and wherein said amplification and enzyme reagents provided to reaction receptacles of said second portion of reaction receptacles in step a) include a primer or a promoter-primer, nucleoside triphosphates, and a nucleic acid polymerase capable of extending the 3′

end of said primer or promoter-primer hybridized to said second target nucleic acid under the temperature conditions of said first heater.

17. The method of claim 16, wherein said temperature conditions are isothermal.

18. The method of claim 1, wherein said detection reagents for detecting said first amplification product include a self-hybridizing probe that detectably hybridizes to said first target sequence or its complement under the temperature conditions of said first heater.

19. The method of claim 18, wherein said probe is a molecular beacon or a molecular torch and includes an interacting label pair.

20. The method of claim 19, wherein said label pair includes a fluorophore and a quencher.

21. The method of claim 1 further comprising simultaneously measuring said signal from each of a plurality of reaction receptacles of said first portion of reaction receptacles.

22. The method of claim 21, wherein, in step b), reaction receptacles of said first portion of reaction receptacles are successively and periodically moved into operative proximity to a plurality of spaced-apart signal measuring devices, each signal measuring device being a fluorescent signal detection device operatively associated with said heater.

23. The method of claim 22, wherein each of said fluorescent signal detection devices is movable relative to said heater.

24. The method of claim 1 further comprising simultaneously measuring multiple signals in reaction receptacles of said first portion of reaction receptacles, each of said multiple signals being associated with a different amplification product.

25. The method of claim 1 further comprising the automated step of exposing sample material present in each of said reaction receptacles to reagents and conditions sufficient to remove amplification inhibitors from said reaction receptacles prior to step a).

26. The method of claim 25, wherein said exposing step includes:

providing a solid support material to each said reaction receptacle, said solid support material being capable of binding said first or second target nucleic acid;

isolating said solid support material within each said reaction receptacle;

removing at least a portion of said sample material from each said reaction receptacle; and

washing said solid support material with a wash solution.

27. The method of claim 26, wherein said solid support material is a magnetically responsive particle and said isolating step is performed with one or more magnets being positioned adjacent each said reaction receptacle.

28. The method of claim 25 further comprising transporting said reaction receptacles to said first heater using an automated reaction receptacle transport system after said exposing step.

29. The method of claim 28, wherein said transport system comprises a rotatable transport mechanism.

30. The method of claim 1, wherein each said reaction receptacle is one of a plurality of integrally formed reaction receptacles.

31. The method of claim 26, wherein the steps of this method are all performed within a housing of a self-contained analyzer unit.