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Systems and methods for multiplex analysis of PCR in real time

  • US 9,745,620 B2
  • Filed: 10/16/2015
  • Issued: 08/29/2017
  • Est. Priority Date: 12/13/2006
  • Status: Active Grant
First Claim
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1. A method of detecting a target molecule in a sample comprising:

  • (a) forming a capture sandwich complex by binding a target molecule, if present in a sample, with (i) a capture antibody, and (ii) a detection antibody, wherein the capture antibody is linked to a magnetic particle and the detection antibody is linked to a detection nucleic acid sequence;

    (b) applying a magnetic field to a surface of a chamber containing the capture antibody linked to the magnetic particle to draw the magnetic particle to the surface in order to immobilize the capture sandwich complex on the surface of the chamber, wherein the magnetic field is applied before or after the capture sandwich complex is formed;

    (c) while the capture sandwich complex is immobilized on the surface of the chamber, washing the chamber to remove excess detection antibody from the chamber;

    (d) introducing into the chamber a primer pair for priming amplification of the detection nucleic acid sequence, a labeling agent, and a probe complementary to an amplification product of the primer pair and the detection nucleic acid sequence, wherein the probe is immobilized on an encoded magnetic bead such that the identity of the probe is known from the encoded magnetic bead on which it is immobilized;

    (e) performing an amplification cycle to form a labeled amplification product of the detection nucleic acid sequence amplified with the primer pair;

    (f) hybridizing the labeled amplification product to the probe immobilized on the encoded magnetic bead; and

    (g) detecting the target molecule, if present in the sample, by detecting a signal from the encoded magnetic bead and a signal from the labeled amplification product.

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