SYSTEMS AND METHODS FOR MULTIPLEX ANALYSIS OF PCR IN REAL TIME

• US 20170175175A1
• Filed: 03/03/2017
• Published: 06/22/2017
• Est. Priority Date: 12/13/2006
• Status: Active Grant

• Current Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Original Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Inventors: WHITMAN, Douglas F., COLLINS, Charles J.
• Granted Patent: US 10,253,354 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label

Abstract

The present invention provides methods and systems for real-time measurements of PCR with multiplexing capability. Certain embodiments relate to methods and systems that use fluorescently encoded superparamagnetic microspheres for the immobilization of amplification products during the PCR process, and an imaging chamber of a measurement device that is also capable of controllable thermal cycling for assisting the PCR process.

Claims

1-37. -37. (canceled)

38. A method for multiplexed detection of nucleic acid targets in a sample comprising:

(a) combining in a chamber (i) a sample, (ii) a plurality of primer pairs for priming amplification of nucleic acid targets in the sample, wherein at least one primer of each primer pair comprises a label, and (iii) a first plurality of probes complementary to labeled amplification obtained by amplification of the nucleic acid targets by the primer pairs, wherein the probes are immobilized on a plurality of encoded microspheres such that the identity of each probe is known from the encoded microspheres on which it is immobilized;

(b) performing a plurality of amplification cycles to form labeled amplification products for each of the nucleic acid targets amplified with the plurality of primer pairs;

(c) hybridizing the labeled amplification products to the probes immobilized on the encoded microspheres;

(d) during the plurality of amplification cycles, measuring a signal from the encoded microspheres and a signal from the labeled amplification products to the probes immobilized on the encoded microspheres, wherein the encoded particles are immobilized on a surface of the chamber, and wherein an increase in the signal from the labeled amplification products hybridized to the probes immobilized on the encoded microspheres during the plurality of amplification cycles indicates the presence of the target nucleic acids; and

(e) detecting the presence or absence of the nucleic acid targets in the sample based on the presence or absence of the signal from the labeled amplification products hybridized to the probes immobilized on the encoded microspheres.

39. The method of claim 38, wherein the sample comprises between 8 to 60 distinct nucleic acid targets.

40. The method of claim 38, wherein the label is a fluorescent dye.

41. The method of claim 38, wherein the encoded microspheres are encoded with fluorescent dyes.

42. The method of claim 38, wherein the encoded microspheres comprise polystyrene.

43. The method of claim 38, wherein the encoded microspheres are magnetic microspheres and the magnetic microspheres are immobilized on the surface of the chamber by a magnetic field.

44. The method of claim 38, wherein the probes comprise a polymerase blocker at their 3′

ends.

45. A method for multiplexed detection of nucleic acid targets in a sample comprising:

(a) combining in a chamber a sample, a plurality of primer pairs for priming amplification of nucleic acid targets in the sample, a plurality of first probes complementary to the nucleic acid targets and immobilized on a plurality of encoded microspheres such that the identity of each first probe is known from the encoded microspheres on which it is immobilized, and a plurality of second probes complementary to the nucleic acid targets, wherein the second probes (i) are labeled, (ii) are not immobilized on the plurality of encoded microspheres, and (iii) are complementary to different regions of the nucleic acid targets than the first probes;

(b) performing a plurality of amplification cycles to form amplification products for each of the nucleic acid targets amplified with the plurality of primer pairs;

(c) during the plurality of amplification cycles, measuring a signal from the encoded microspheres and a signal from the label of the second probes hybridized to the amplification products that are hybridized to the first probes immobilized on the encoded microspheres, wherein the encoded particles are immobilized on a surface of the chamber during the plurality of amplification cycles; and

(d) detecting the presence of the nucleic acid targets in the sample based on the presence of the signal from the label of the second probes hybridized to the (e) amplification products that are hybridized to the first probes immobilized on the encoded microspheres.

46. The method of claim 45, wherein the sample comprises between 8 to 60 distinct nucleic acid targets.

47. The method of claim 45, wherein the second probes are labeled with a fluorescent dye.

48. The method of claim 45, wherein the second probes are labeled with a quencher and the first probes are labeled with a fluorescent dye.

49. The method of claim 45, wherein the encoded microspheres are encoded with fluorescent dyes.

50. The method of claim 45, wherein the encoded microspheres are magnetic microspheres and the magnetic microspheres are immobilized on the surface of the chamber by a magnetic field.