Methods and systems for multiplex quantitative nucleic acid amplification

• US 10,174,367 B2
• Filed: 10/12/2016
• Issued: 01/08/2019
• Est. Priority Date: 09/10/2015
• Status: Active Grant

• Current Assignee: Insilixa, Inc. (Danaher Corporation)
• Original Assignee: Insilixa, Inc. (Danaher Corporation)
• Inventors: Hassibi, Arjang, Jirage, Kshama, Manickam, Arun, Singh, Rituraj
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/291,747
• Publication Number:
  US 20170101666A1
• Time in Patent Office: 818 Days
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

The present disclosure provides methods, devices and systems that enable simultaneous multiplexing amplification reaction and real-time detection in a single reaction chamber.

Claims

1. A method for assaying at least one template nucleic acid molecule, comprising:

(a) activating a sensor array comprising (i) a substrate comprising a plurality of first probes immobilized to a first pixel, a plurality of second probes immobilized to a second pixel, wherein said first probes are configured to capture an individual primer of a primer set, and wherein said second probes are configured to to capture a control nucleic acid molecule, and (ii) an array of detectors configured to detect at least one first signal from said first pixel and at least one second signal from said second pixel, wherein a difference between said at least one first signal and said at least one second signal over time is indicative of said individual primer binding with an individual probe of said plurality of first probes;

(b) subjecting a reaction mixture to a nucleic acid amplification reaction under conditions sufficient to yield at least one target nucleic acid molecule as an amplification product(s) of said template nucleic acid molecule, wherein said reaction mixture comprises (i) a nucleic acid sample containing or suspected of containing said template nucleic acid molecule, (ii) said primer set, (iii) said control nucleic acid molecule, and (iv) a polymerizing enzyme, wherein said individual primer of said primer set has sequence complementarity with said template nucleic acid molecule;

(c) using said array of detectors to detect said at least one first signal and said at least one second signal at multiple time points during said nucleic acid amplification reaction; and

(d) using said difference between said at least one first signal and said at least one second signal to detect said template nucleic acid molecule.

2. The method of claim 1, wherein said at least one first signal is produced upon binding of said individual probe to said individual primer, and wherein said at least one second signal is produced upon binding of an additional probe of said second probes to said control nucleic acid molecule.

3. The method of claim 1, wherein said control nucleic acid molecule is not amplified in said amplification reaction.

4. The method of claim 1, wherein said reaction mixture comprises a plurality of template nucleic acid molecules, and wherein said first probes specifically bind to a plurality of target nucleic molecules as amplification products of said plurality of said template nucleic acid molecules.

5. The method of claim 1, wherein said primer set comprises a plurality of individual primers having different nucleic acid sequences, and wherein said first probes are configured to specifically bind to said plurality of said individual primers.

6. The method of claim 1, wherein said reaction mixture is provided in a reaction chamber configured to retain said reaction mixture and permit said first and second probes to bind to said individual primer and said control nucleic acid molecule.

7. The method of claim 1, further comprising correlating said at least one first signal detected at multiple time points with an initial concentration of said at least one template nucleic acid molecule by analyzing a binding rate of said probes with said individual primer from said primer set.

8. The method of claim 1, wherein said first probes or said second probes are oligonucleotides.

9. The method of claim 1, wherein said sensor array comprises at least about 100 integrated sensors.

10. The method of claim 1, wherein said at least one first signal is a first optical signal that is indicative of a first interaction between a first energy acceptor and a first energy donor associated with said individual primer and said individual probe, and wherein said at least one second signal is a second optical signal that is indicative of a second interaction between a second energy acceptor and a second energy donor associated with said control nucleic acid molecule and an additional probe of said second probes.

11. The method of claim 10, wherein said first energy acceptor is coupled to said individual primer, and wherein said second energy acceptor is coupled to said control nucleic acid molecule.

12. The method of claim 10, wherein said first energy acceptor is coupled to said target nucleic acid molecule.

13. The method of claim 10, wherein said first energy acceptor is a first quencher, and wherein said second energy acceptor is a second quencher.

14. The method of claim 10, wherein said first energy donor is a first fluorophore, and wherein said second energy donor is a second fluorophore.

15. The method of claim 10, wherein said first energy donor is coupled to said first probe, and wherein said second energy donor is coupled to said second probe.

16. The method of claim 1, wherein said target nucleic acid molecule is detected at a sensitivity of at least about 90%.

17. The method of claim 1, wherein said at least one first signal is detected while said reaction mixture comprising said target nucleic acid molecule is in fluid contact with said sensor array.

18. A system for assaying at least one template nucleic acid molecule, comprising:

(a) a reaction chamber comprising a reaction mixture, wherein said reaction mixture comprises (i) a nucleic acid sample containing or suspected of containing said template nucleic acid molecule, (ii) a primer set comprising an individual primer, (iii) a control nucleic acid molecule, and (iv) a polymerizing enzyme, wherein said individual primer of said primer set has sequence complementarity with said template nucleic acid molecule, wherein said reaction chamber comprising said reaction mixture is configured to facilitate a nucleic acid amplification reaction with said reaction mixture under conditions sufficient to yield at least one target nucleic acid molecule as an amplification product(s) of said template nucleic acid molecule, wherein said nucleic acid amplification reaction does not yield any amplification product of said control nucleic acid;

(b) a sensor array comprising (i) a substrate comprising a plurality of first probes immobilized to a first pixel, a plurality of second probes immobilized to a second pixel, wherein said first probes are configured to capture said individual primer of said primer set, and wherein said second probes are configured to capture said control nucleic acid molecule, and (ii) an array of detectors configured to detect at least one first signal from said first pixel and at least one second signal from said second pixel, wherein a difference between said at least one first signal and said at least one second signal over time is indicative of said individual primer binding with an individual probe of said plurality of first probes; and

(c) a computer processor coupled to said sensor array and programmed to (i) subject said reaction mixture to said nucleic acid amplification reaction, and (ii) detect said at least one first signal and said at least one second signal at multiple time points during said nucleic acid amplification reaction.

19. The system of claim 18, wherein said computer processor is programmed to detect said template nucleic acid molecule using said difference between said at least one first signal and said at least one second signal.

20. The system of claim 18, wherein said reaction mixture comprises a plurality of template nucleic acid molecules, and wherein said first probes specifically bind to a plurality of target nucleic molecules as amplification products of said plurality of said template nucleic acid molecules.

21. The system of claim 18, wherein said primer set comprises a plurality of individual primers having different nucleic acid sequences, and wherein said first probes are configured to specifically bind to said plurality of said individual primers.

22. The system of claim 18, wherein said array of detectors comprises an optical detector.

23. The system of claim 22, wherein said at least one first signal is a first optical signal that is indicative of a first interaction between a first energy acceptor and a first energy donor associated with said individual primer and said individual probe, and wherein said at least one second signal is a second optical signal that is indicative of a second interaction between a second energy acceptor and a second energy donor associated with said control nucleic acid molecule and an additional probe of said second probes.

24. The system of claim 22, wherein said optical detector comprises a complementary metal-oxide semiconductor device.

25. The system of claim 18, wherein said array of detectors comprises an electrical detector.

26. The system of claim 25, wherein said electrical detector comprises a complementary metal-oxide semiconductor device.

27. The system of claim 18, wherein said sensor array comprises at least about 100 integrated sensors.