Systems and methods for multiplex analysis of PCR in real time

摘要

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.

主权项

1. A method of amplifying and detecting a plurality of nucleic acid targets in a sample comprising:
(a) combining in a chamber a sample comprising the plurality of nucleic acid targets, a plurality of primer pairs for priming amplification of the plurality of nucleic acid targets, a labeling agent, and a first plurality of probes complementary to the plurality of nucleic acid targets, wherein the probes are immobilized on a plurality of encoded particles such that the identity of each probe is known from the encoded particle on which it is immobilized; (b) performing an amplification cycle to form labeled amplification products for each of the plurality of nucleic acid targets amplified with the plurality of primer pairs; (c) hybridizing the labeled amplification products to the probes immobilized on the encoded particles; (d) attracting the encoded particles to a surface of the chamber by gravity; (e) detecting a signal from the encoded particles and a signal from the labeled amplification products hybridized to the probes immobilized on the encoded particles while the encoded particles and the labeled amplification products hybridized to the probes immobilized on the encoded particles are on the surface of the chamber; (f) dispersing the encoded particles from the surface of the chamber; and (g) repeating steps (b) through (f) at least once; wherein the plurality of nucleic acid targets in the sample are amplified and detected.