Method for continuous mode processing of multiple reaction receptacles in a real-time amplification assay
View Patent ↗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.
1. A method for processing the contents of multiple reaction receptacles to quantitatively determine the amount of a target nucleic acid present in each of said reaction receptacles, said method comprising the automated steps of:
a) successively providing said reaction receptacles to a temperature-controlled chamber of an incubator having a housing defining said temperature-controlled chamber, said housing including one or more closable receptacle access openings for moving the reaction receptacles into and out of said incubator, each reaction receptacle being provided with sample material and reagents sufficient to amplify and detect the presence of a target sequence, or its complement, contained within a target nucleic acid under the temperature conditions of said incubator, wherein successively providing the reaction receptacles to said the temperature-controlled chamber comprises, for each of the reaction receptacles provided, opening one of said receptacle access openings and moving the reaction receptacle through the opened receptacle access opening and into the temperature-controlled chamber and then closing the receptacle access opening, thereby permitting ambient light from within the analyzer to enter the temperature-controlled chamber of the incubator through the opened receptacle access opening;
b) in said incubator, periodically measuring the amount of a signal associated with the presence of an amplification product containing said target sequence or its complement in each reaction receptacle provided to said incubator in accordance with step a);
c) quantifying the amount of said target nucleic acid present in each reaction receptacle based upon said measurements obtained in step b); and
d) successively removing reaction receptacles from the temperature-controlled chamber, wherein successively removing the reaction receptacles from said temperature-controlled chamber comprises, for each reaction receptacle removed from said temperature-controlled chamber, opening one of said receptacle access openings and moving the reaction receptacle through the opened receptacle access opening and out of said temperature-controlled chamber of said incubator and then closing the receptacle access opening, thereby permitting ambient light from within the analyzer to enter the temperature-controlled chamber of the incubator through the opened receptacle access opening,
wherein reaction receptacles are being successively provided to said incubator in accordance with step a) as the contents of reaction receptacles previously provided to said incubator are being processed in accordance with steps b)-c).
2. The method of claim 1 , wherein said reaction receptacles are provided to said incubator using an automated reaction receptacle transport system.
3. The method of claim 2 , wherein said reaction receptacles having contents processed in accordance with steps a)-c) are successively removed from said incubator as said reaction receptacles having contents for processing in accordance with steps a)-c) are successively provided to said incubator using said automated reaction receptacle transport system.
4. The method of claim 2 , wherein said automated reaction receptacle transport system includes a first rotatable transport mechanism for providing said reaction receptacles to said incubator.
5. The method of claim 4 , wherein said automated reaction receptacle transport system includes a second rotatable transport mechanism for removing said reaction receptacles from said incubator.
6. The method of claim 4 , wherein said incubator includes a reaction receptacle carrier rotatable about an axis of rotation.
7. The method of claim 1 , wherein said housing of said incubator includes one or more doors for receiving and removing said reaction receptacles from said incubator.
8. The method of claim 7 , wherein said incubator includes a rotatable reaction receptacle carrier contained within said housing for receiving and moving reaction receptacles within said incubator.
9. The method of claim 1 , wherein said reagents are provided to said reaction receptacles after said reaction receptacles are provided to said incubator.
10. The method of claim 1 , wherein said reagents 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 target nucleic acid under the temperature conditions of said incubator.
11. The method of claim 1 , wherein said temperature conditions are isothermal.
12. The method of claim 1 , wherein said reagents include a self-hybridizing, detectably labeled probe that binds to said target sequence or its complement under the temperature conditions of said incubator, thereby permitting said probe to emit said signal.
13. The method of claim 12 , wherein said probe includes an interacting label pair.
14. The method of claim 13 , wherein said label pair includes a fluorescent moiety and a quencher moiety.
15. The method of claim 1 further comprising simultaneously measuring said signal from each of a plurality of said reaction receptacles.
16. The method of claim 15 , wherein said signal is measured with a plurality of spaced-apart fluorescent signal detection devices operatively associated with said incubator.
17. The method of claim 1 further comprising simultaneously measuring a different signal in each of a plurality of said reaction receptacles, each of said different signals being associated with a different amplification product.
18. The method of claim 17 , wherein said different signals are measured with a plurality of spaced-apart fluorescent signal detection devices operatively associated with said incubator and movable relative thereto.
19. 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).
20. The method of claim 19 , wherein said exposing step includes: providing a solid support material to each reaction receptacle, said solid support material being capable of directly or indirectly binding said target nucleic acid; isolating said solid support material within each reaction receptacle; removing at least a portion of said sample material from each reaction receptacle; and washing said solid support material with a wash solution.
21. The method of claim 20 , wherein said solid support material is a magnetically responsive particle and said isolating step is performed with magnets positioned adjacent said reaction receptacles.
22. The method of claim 19 further comprising transporting said reaction receptacles to said incubator using an automated reaction receptacle transport system after said exposing step.
23. The method of claim 22 , wherein said automated reaction receptacle transport system comprises a rotatable transport mechanism.
24. The method of claim 1 , wherein each reaction receptacle is one of a plurality of integrally formed reaction receptacles.
25. The method of claim 1 , wherein the steps of the method are performed within a self-contained analyzer unit.
26. The method of claim 1 , wherein said measuring is performed by using one or more signal measuring devices, each said signal measuring device having a portion thereof located outside said housing.